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Strengthening of the MagBioVin Research and Innovation Team for Development of Novel Approaches for Tumour Therapy based on Nanostructured Materials

Final Report Summary - MAGBIOVIN (Strengthening of the MagBioVin Research and Innovation Team for Development of Novel Approaches for Tumour Therapy based on Nanostructured Materials)

Executive Summary:
Through realization of the MagBioVin project, the Institute of Nuclear Sciences “Vinca”, University of Belgrade (Serbia), received important community support within the FP7-ERA Chairs Program to upgrade its human and experimental capacities dedicated to the highly specialized and interdisciplinary research on activated magnetic nanoparticles (MNP) and radionuclide labeled magnetic nanoparticles for application in biosciences, pharmacy and medicine. By following this general direction a set of comprehensive actions, subdivided into six Work packages, were executed to achieve the following Project objectives:

o Reinforcement of the human resources by employment of the peer scientist in the field (ERA Chair holder) and several prominent experts from different areas that will make the core of the MagBioVin multidisciplinary team;
o Upgrading of experimental capacities by the provision of the specialized equipment for targeted research topics;
o Adoption of up-to-date know-how through visits and short stays of expert scientists for knowledge transfer through lectures and practical demonstrations;
o Training of team members in cutting-edge research techniques in reputable research institutions from EU countries;
o Improved regional and international networking for reinforcement of international cooperation and integration in European Research Area (ERA);
o Dissemination of project progress and achieved results to different target groups: to scientific community by participation in scientific meetings of high level and publication of papers; to students and young researchers by lectures and seminars given at different universities in the country; to stakeholders such as pharmaceutical companies and medical centers by presentations at national/international meetings; to broader public by printed materials written in popular manner.

The Project anticipated the key role of the ERA Chair holder in improvement of organizational structure and research excellence, as well as in regional/international networking and communication with stakeholders. Besides considerable experimental facility improvement, a key factor in the upgrade of the research excellence was the assembly of the ERA Chair team whose core consisted of prominent experts in the highly specialized research areas which were underdeveloped before. Additional knowledge reinforcement was done through organization of seven dedicated workshops led by EU experts in the field, six extensive training programs for MagBioVin team members held in eminent research centers from EU, as well as by lectures given by peer experts in the field during their visits to "Vinca". Increase excellence resulted in active participation of team members at highly ranked international scientific meetings and publication of research papers in peer-reviewed journals. Additionally, foundation of the Center of Excellence for Magnetic and Radiolabeled Nanomaterials as a new constitutive unit of the "Vinca" Institute has been completed, while four recruited researchers (including ERA Chair holder) and project manager from the project team obtained positions in "Vinca" after the end of the project.

By intensifying both regional and international networking MagBioVin team increased its collaborative capacities and visibility so that more than twenty joint proposals for research grants at international level were prepared and submitted. Eleven of these projects obtained funding and their realization is currently in progress.

All Project activities resulted in building a solid foundations for a final goal: to raise both human and experimental capacities of the “Vinca” Institute up to the level that ensures visible and sustainable scientific and technological impact within ERA, and with the increased competitiveness comparable to that of the prominent research institutions in EU.

Project Context and Objectives:
The motivation for MagBioVin project was the need of the Institute of Nuclear Sciences “Vinca”, University of Belgrade (Serbia, Belgrade), to obtain community support for reinforcement of its S&T and innovative capacities (both human and experimental) in the field of the highly specialized research on activated magnetic nanoparticles (MNP) and radionuclide labeled magnetic nanoparticles for application in biosciences, pharmacy and medicine.

The "Vinca" Institute (http://www.vin.bg.ac.rs/index.php/en/) was founded in 1948, and nowadays it is one of the most prestigious scientific institutions in the country, and among largest scientific centers in South East Europe. It consists of 13 laboratories and seven research centers with the total number of employees close to 800, including more than 300 PhDs and MSc, and more than 100 PhD students. During its 70 years of tradition the Institute has gradually changed its activities from mainly nuclear research program to today's multidisciplinary activities concerning theoretical and condensed matter physics, material sciences, nuclear physics, atomic physics, physical chemistry, radiation chemistry, radiobiology, molecular genetics, endocrinology, medicine, electronics, computer sciences, etc. Due to diversity in research fields and communication among scientists of different profiles, "Vinca" constantly cultures multidisciplinary character of its research programs.

The orientation to applications of MNP in medicine is the result of a very broad initiative related to the Institute's strategic development which was guided by both Institute's Scientific Council and Management Board. This initiative comprehended all Institute's laboratories which cover broad scientific areas ranging from physics, chemistry and material sciences to biology and health. The outcome of detailed analysis was the document on the Institute strategic development which showed that the multidisciplinary research comprising nanotechnology, biology and health had the highest opportunities to mobilize majority of researchers and attain success. In addition, such an orientation complies with the thematic priorities' domains of the EC FP7 (Themes: 1. Health, 4. Nanosciences, Nanotechnologies, Materials and new Production Technologies), and in that sense it has the highest feasibility for the efficient integration of the "Vinca" Institute in the European Research Area (ERA).

In order to identify research groups capable to achieve the above stated strategic orientation SWOT analysis was done for several Institute's most reputable departments working in the fields of nanoscience, radiopharmaceuticals, genetics, and molecular biology and pharmacy. Consequently, Institute's Management Board supported formation of multidisciplinary MagBioVin team for research and innovation in the field of nanomaterials and their applications in biomedicine. Team comprehends six groups covering different areas of research and development:
- Group for the synthesis and functionalization of MNPs;
- Group for material characterization and computational modelling;
- Group for application of nanomaterials in hyperthermia;
- Group for application of nanomaterials in radionulides therapy;
- Group for biological response to applications of MNPs in therapy (cytotoxicity,
genomics and transcriptomics);
- Group for IP, technology transfer and promotion.

Human and experimental resources of the MagBioVin team were considerable, since it consisted of researchers with the proven scientific records and laboratories well equipped for the planned research. However, several weaknesses listed below were identified, and they needed to be overcome in order to achieve feasible and productive outcomes:
- Deficiency of outstanding researchers with the experience in managing complex multidisciplinary research team;
- Deficiency in human resources, especially concerning highly specialized researchers, mostly due to severe brain-drain problems;
- Lack of specialized equipment - the diverse research areas of the "Vinca" Institute are covered by relatively adequate equipment of the general purpose, however a lack of state-of-the art equipment aimed to highly specialized research was evident;
- Insufficient involvement in contemporary, state of the art trends in nano-science, which is a direct consequence of unsatisfactory integration level in ERA;
- A relatively modest Institute's visibility at the international level;
- Unsatisfactory interaction and knowledge transfer to stakeholders - at the current stage, the Institute's relationship with end users is mostly based on providing specific services, rather than participation in active development, with patents and innovations as end results.

For overcoming the above listed weaknesses a wider community support was necessary, and ERA Chairs call as a new FP7-CSA instrument provided adequate support for such a goal. Consequently, MagBioVin project was put together to address all the weak points identified, and corresponding set of main Project objectives was formulated:
(i) Reinforcement of the human resources by employment of the peer scientist in the field (ERA Chair holder) and several prominent experts from different areas that will make the core of the MagBioVin multidisciplinary team;
(ii) Upgrading of experimental capacities by the provision of the specialized equipment for targeted research topics;
(iii) Adoption of up-to-date know-how through visits and short stays of expert scientists for knowledge transfer through lectures and practical demonstrations;
(iv) Training of team members in cutting-edge research techniques in reputable research institutions from EU countries;
(v) Improved regional and international networking for reinforcement of international cooperation and integration in European Research Area (ERA);
(vi) Dissemination of project progress and achieved results to different target groups: to scientific community by participation in scientific meetings of high level and publication of papers; to students and young researchers by lectures and seminars given at different universities in the country; to stakeholders such as pharmaceutical companies and medical centers by presentations at national/international meetings; to broader public by printed materials written in popular manner.

In accordance to the call, the Project anticipated the key role of the ERA Chair holder in improvement of organizational structure and research excellence, as well as in regional/international networking and communication with stakeholders. Besides considerable experimental facility improvement, a key factor in the upgrade of the research excellence was the assembly of the ERA Chair team whose core consisted of prominent experts in the highly specialized research areas which were underdeveloped before.

Important role in the project is also anticipated from the strategic partner institutions which are highly developed research centers from four EU countries (Spain, France, Slovenia, and Slovakia). They all expressed their commitments in providing logistic support to the project by delegating their researchers as lecturers/demonstrators for workshops in “Vinca”, by hosting MagBioVin staff in their laboratories for training purposes, and by participating in project Steering Committee.

The activities foreseen for realization of Project goals are subdivided into six Workpackages which are complementary and closely interconnected:
WP1: Strengthening of MagBioVin human resources;
WP2: Provision of experimental facilities;
WP3: Adoption of Advanced Methodologies and Know-How;
WP4: Outgoing mobility and reinforcement of international cooperation;
WP5: Dissemination and Promotion;
WP6: Project management.

The central point of WP1 is appointment of the peer scientist with the proven record in scientific excellence, fund raising and management capabilities, for the role of the ERA Chair holder. Under his/her supervision the team will be additionally reinforced by hiring of two experienced scientists and three Post Docs, each of them with the expertise in the previously underdeveloped fields. In addition, recruitment of three PhD students will provide basis for the sustainability of the research after the project lifetime.

WP2 provides the necessary up-to-date experimental facilities for different research stages, from MNPs synthesis, their structural and morphological characterization, to magnetic hyperthermia investigation (in vitro and in vivo), and biological/biochemical aspects of the research (biodistribution of activated and radiolabeled MNPS, cell and tissue manipulation and investigation, etc.).

WP3 provides adoption of know-how and development of the state-of-the-art methodologies/techniques currently used in cutting-edge researches on application of MNPs in biomedicine. This will be achieved by knowledge transfer through workshops, lectures and demonstrations given either by expert scientist during their short stays in “Vinca” under the invitation of the ERA Chair, or by newly recruited MagBioVin staff. The elevated level of knowledge and skills will enable the foundation of the new Center of Excellence in the “Vinca” Institute.

The goal of WP4 is to enhance the mobility of MagBioVin researchers and to facilitate the exchange of know-how and experience mainly between MagBioVin and strategic partner institutions. Six training programs are anticipated in order to allow for significant enhancement of experimental and theoretical skills of the MagBioVin staff by short stays and active participation in ongoing research activities of partner institutions. Projected outcomes of this task is acquiring mission critical technical experience, adoption of know-how and good practices, as well as establishing a firm basis for long-term collaborative work and future joint project proposals with strategic partners. Besides already defined partners, new contacts established under the guidance of Era Chair and recruited experience scientist will enable initiating and preparation of joint proposals for different funding opportunities in order to expand and reinforce international cooperation.

WP5 deals with both dissemination of project results and its promotion to different target groups. Dissemination of knowledge and scientific results is aimed to national and international scientific community through publication of papers and participation at conferences, workshops and seminars. Dissemination of project goals and achievements to stakeholders comprehends universities, medical centers and pharmaceutical companies, while promotional activities are mostly aimed to policy making structures and broader public in order to raise awareness of the possibilities and development of the local scientific institutions.

Management structure and its role is summarized in WP6 with the general goal to provide efficient and timely execution of all activities planned within other WPs. Project management consists of Management Board that is composed of five WP leaders, Project coordinator and ERA Chair, and Steering Committee whose members are representatives from Strategic Partner institutions. Administrative tasks are predominantly under the responsibility of Project Coordinator and WP leaders, while scientific management is under the ERA Chair supervision. Control and advisory role is assign to Steering Committee due to a large experience of its members in managements of complex multidisciplinary projects. It should be emphasized that the described management structure prove to be efficient since majority of the project tasks have been successfully accomplished in spite of many difficulties that emerged during the project lifetime. Some of these difficulties originate from uncoordinated legislative and administrative procedures in Serbia and EU. In particular, this concerns custom and tax rules, procedures for employment of foreign citizens, and public procurement procedures, which all caused delays and additional engagement of the project staff. Another difficulty concerns recruitment of the ERA Chair holder and his/her team due to a low interest of qualified candidates, in spite of attractive job offers in respect to salaries, work conditions and benefits. Candidates for PhD positions were also difficult to recruit, most probably due to a brain-drain effect that affects Serbian society for quite some time.

Project Results:
The foregrounds of the realized MagBioVin project can be divided into two categories:

- Achievements made in project realization in terms of a supporting action program - these imply successful development of the state-of-art research capacities regarding both human and experimental resources, and
- Achievements made in the research process, in terms of raised quality and widening of research fields.

I. Improvement of human and experimental resources

1. Reinforcement of human capacities

The prerequisite for the project realization, and at the same time, the backbone for achieving the desired results, was recruitment and formation of an ERA Chair team. Considering complex geo-political circumstances within western Balkan region, MagBioVin management board was coerced to invest a great effort to find and select the best among suitable candidates for open ERA Chair team positions. Although not all of the recruited staff was active at the same time and over the whole project period, core of the ERA Chair team has been always preserved functional, even during the time of ERA Chair holder change. Over the course of the project realization, two ERA Chair holders, three Senior Scientists, five Post-docs, two PhD students and one project manager were recruited:

- ERA Chair holder: Prof. Dr Andrzej Szytula (Professor Emeritus, Physics, stepped down from the position due to health issues and replaced by Dr Zeljko Prijovic during 2016.)
- Dr Davide Peddis (Senior Scientist, physical chemistry)
- Dr Zeljko Prijovic (Senior Scientist, biochemistry, took the position of ERA CHAIR during 2016.)
- Dr Biljana Dojcinovic (Senior Scientist, analytical chemistry)
- Dr Erzsebet Illes (Post-doc, inorganic chemistry)
- Dr Nikola Knezevic (Post-doc, organic/inorganic chemistry)
- Dr Dalibor Stankovic (Post-doc, chemistry/biochemistry)
- Dr Martin Fabian (Post-doc, physical chemistry)
- Dr Jose Mariano (Post-doc, physics)
- BSc Milos Ognjanovic (PhD student, physical chemistry)
- BSc Zorana Milanovic (PhD student, veterinary medicine)
- BSc Luka Ivancic (project manager)

It should be stressed out that the most important accomplishment concerning human resource strengthening is, in fact, the preservation of MagBioVin team beyond the project duration. Five members of the above mentioned recruited staff, including the ERA Chair holder, continued their scientific careers at the "Vinca" Institute after the end of project realization: Dr Zeljko Prijovic, Dr Dalibor Stankovic, BSc Milos Ognjanovic (PhD student), Dr Zorana Milanovic (completed her PhD studies) and Luka Ivancic (as administrative manager).

Thus the important basis of advanced research structure in fields of cancer combat by local radiotherapy and hyperthermia that was developed and supported by the MagBioVin project is successfully sustained. Though ensuring the sustainability of the project results was one of important project goals, it can be concluded that the extent to which the MagBioVin team is preserved and its compliance with current research funding programs within the "Vinca" Institute goes beyond initial expectations.
2. Raising the excellence of the MAGBIOVIN team

A) Trainings in partner institutions

Besides the recruitment of the ERA Chair team, human recourses were also substantially strengthened via adoption of new methodologies and know-how. The knowledge transfer towards MagBioVin staff was achieved by using the advanced expertise of ERA Chair holder and new recruited staff, through visits of expert scientists, and by series of trainings in advanced methodologies performed at partner institutions, enabled through MagBioVin support for outgoing mobility. By increasing the quality of scientific production and the technological development as well as investing in advancement of knowledge we have assured the research sustainability. Advances have been made through the increasing of the Outgoing mobility and international cooperation.

During the five years project period, several trainings of MagBioVin researchers in the reputable EU partner institutions were organized. They were aimed to increase the mobility of MagBioVin researchers (research staff, young scientists, and PhD students) and to facilitate the exchange of know-how and experience between MagBioVin and EU strategic partner institutions. The adopted knowledge and best practices were successfully implemented in the MagBioVin research activities through setting of new methods, protocols and procedures.

Thirteen trainings were successfully performed covering variety of the research topics in the field of biochemistry and medicine, biology, physics and material science. Eleven young researchers, members of the MagBioVin team, were successfully trained. Full description of all realized trainings can be found in three Periodical Reports, while few examples of the good practice implementation are given below:

A.1) Training program of MagBioVin PhD student Aleksandar Vukadinovic, organized in collaboration with University of Patras (Greece). This was particularly aimed at enhancement of experimental skills in biochemistry and to gain necessary routine. Aleksandar Vukadinovic was send to the Laboratory of School of Medicine at the University of Patras, the well established department in the area of cancer research, and his training covered tissue culture techniques including preservation in liquid nitrogen and re-viving (thawing), culture, propagation, harvesting, counting etc. Inoculation of the cells to generate s.c. human tumor xenografts in immune-compromised mice was practiced on NOD-SCID mice and using LS174T human colon cancer. For that purpose, preparation of animals and cells for the injection, administration of the cells, maintenance of the treated animals and tumor growth monitoring are practiced.

Treatment of the tumor-bearing animals with antitumor compounds administered via tail vein, i.p and s.c were demonstrated. Further, the blood collection from tail vein puncture and preparation of the samples for analysis of the metabolites by SPE-HPLC were demonstrated. For practical purpose, a collection of urine from treated animals in metabolic cage and isolation of the metabolites by preparative HPLC was practiced. Additionally, the tumor excision by surgery, embedding in OCT and thin sectioning by cryotome, followed by Imune-Histochemistry (IHC) and enzyme-Histochemistry (EHC) were performed. Altogether, this course exposed the young scientist to all the skills and procedures he needed in further work in biochemistry.

A.2) Laboratory for endocrinology of Dr Ioannis Hampeios and Biochemistry department of School of Medicine, University of Patras gave significant support for development of “bio” part of the MagBioVin project. To continue the research and achieve the goals as defined, it was necessary to strengthen ability of the project team to design and perform biology-based experiments (tissue culture, animal models of tumor etc.) as well as to process and analyze the cells and tissues (immune-histochemistry, enzyme-histochemistry, tissue section etc.). As partners Laboratory was well equipped and maintained Vivarium for housing rodents including immune-compromised mice as well as tissue culture room with bank of human cancer cell lines which were of interest to MagBioVin, it represented a place with excellent conditions for training of the project team members who will perform the experiments, allowing them to gain in-hands experience in the techniques. For that purpose, PhD student Aleksandar Vukadinovic and Post Doc Dr. Dalibor Stankovic participated in the training on tissue culture techniques, tumor inoculation, animal handling, tumor removal, tissue sectioning and immuno-histochemical techniques. The participants took part in the experiments, performing the techniques by themselves under guidance of supervisor. Young MagBioVin researchers gained enough hand-in experience to be able to repeat them in the "Vinca" Institute and train other members as necessary.

A.3) Two members of the MagBioVin team, BSc Milos Ognjanovic and Dr Dalibor Stankovic participated in the trainings in Institute of Materials Science of Madrid. Their supervisor was Dr Maria del Puerto Morales, head of the Materials for Health Group, integrated in the Energy, Environment and Health Department. This group is devoted to a search for new protocols regarding the synthesis, microstructure investigation and application of nanomaterials. The activities cover four main fields: preparation of nanomaterials – primarily nanoparticles, modification of nanomaterials by doping or coating, preparation of colloidal suspensions, and characterization of these materials. The trainings were devoted to different research topics such as: synthesis of nanomaterials by using polyol method and thermal decomposition method, process of ligand exchange and synthesis of nanocomposites using hydrothermal microwave route. Training also included various characterization techniques (TEM, XRD, and VSM) in order to examine morphological, microstructural and magnetic properties of prepared samples. Based on experience gained during his visits to ICCM, M. Ognjanovic was able to perform synthesis of iron oxide nanoparticles in order to get biocompatible samples with high specific absorption rate (SAR) value, that were further used at the Vinca Institute as carriers for radiopharmaceuticals for treatments of the mice tumours, as well as for magnetic hyperthermia experiments. Dr Dalibor Stankovic had training on the synthesis procedure for the new carbon-based nanocomposite by using previously synthesised magnetic nanoparticles. Flower-like iron oxide nanoparticles were mixed together with graphene oxide in microwave reactor in order to get nanocomposite decorated nanoparticles on the top of graphene oxide sheets. These kind of magnetic nanoparticles appear to be the most suitable for hyperthermia experiments as they showed significantly increased SAR in comparison to iron oxide nanoparticles synthesized by other methods.

A.4) BSc. Marko Boskovic completed his training in the College of Material Science and Engineering, University of Technology, Beijing, China in the group of Dr.Yue Ming from College of Material Science and Engineering. The group activities are focused on research of modern materials for permanent magnets. Especially, they are skilled in and equipped for synthesis and all-round characterization of magnetic materials. During the stay M. Boskovic was engaged in working with VSM magnetometer, TE microscope, and magnetic field mapping device. After the training in Beijing, M. Boskovic implemented mapping technique during the construction of Faraday balance in the Institute of Nuclear Sciences Vinca.

A.5) Dr Ana Mrakovic stayed at Norwegian University of Science and Technology (NTNU), Trondheim, Norway in order to learn about the advanced synthesis routes for magnetic nanoparticles. During her stay, three different lines of synthesis of ferrite nanoparticles (MFe2O4; M = Fe2+, Co2+, Ni2+) were initiated based on the thermal decomposition method of organometallic precursors in high boiling solvents. The synthesized samples were treated with long chain polymers in order to encapsulate them in biocompatible matrix suitable for further functionalization. Samples were used later as hyperthermia agents in Institute of Nuclear Science Vinca.

A.6) MagBioVin team members Dr. Maja Zivkovic and Dr Aleksandra Stankovic (both molecular biologists) participated at 7th Sardinian international summer school in Calgary, Italy, where they attended practical trainings in the field of identification of the genetic bases of human diseases, gained knowledge in software platforms for analysing sequencing and microarray results and analyzed specific sub-cellular populations in biological samples of interest to research field covered by MagBioVin project support. The school was aimed to the transfer of knowledge on theory and practice of state-of-the-art approaches towards the identification of the genetic bases of human diseases, particularly related to complex and monogenic traits. Covered topics included: Genetics of Autoimmunity, B cell and T cell Receptors analysis, Whole genome, RNA and Single Cell Sequencing, Microbiome and Metagenome analysis, Population Genetics and Genetics of Aging. By participation in the summer school MagBioVin researchers gained the latest scientific and methodological knowledge in the field of genomics and transcriptomics in complex diseases, as well as crucial software platforms for analyzing sequencing and microarray results. An important experience has been gained for applying the same methods at the Institute Vinca, in a Department of radiobiology and molecular genetics that are relevant to the needs of the MagBioVin project.

B) Trainings at workshops organized in MagBioVin facilities at the “Vinca”Institute

Trainings of the MagBioVin researchers were also organized in the "Vinca" Institute through eight dedicated workshops that have covered the cutting-edge methodologies currently in use in the area of MNPs application in biomedicine. The lectures and practical exercises during the MAGBIOVIN workshops were conducted by internationally recognized experts from Project partner organizations, National Research Centres and EU Institutions. Given lectures and discussions initiated afterwards gave MagBioVin researchers the opportunity to gain additional knowledge through the sharing of experiences with eminent experts. However, the most important benefit provided by MagBioVin workshops appeared to be the practical knowledge adopted from colleagues that were more experienced in the specific research fields.
Workshops covered specific topics from physics, chemistry of materials, biochemistry and biomedicine in order to strengthen required multidisciplinary approach. Participation of whole MagBioVin team in the series of workshops resulted in better understanding of ‘whole picture’ as well as problems that could arise during realization of specific project tasks. Finally, all of the organized workshops were open at least partially to scientific public, which increased visibility of MagBioVin team excellence and awareness of its scientific goals. The number of 43 lecturers was rather fascinating with approximately 40 participants per each organized event. Short description of the several workshops that significantly raised MagBioVin team expertise and improved experimental skills is given below:

B.1) Workshop devoted to the "Advanced Synthesis Routes for MNPs" organized primarily to educate MagBioVin early stage researchers working in the field of developing of synthesis methods for nanoparticle production as well as nanoparticle structural characterization. Other participants were not just MagBioVin team members but also some researchers from the “Vinca” Institute interested in the similar topics. Three guest researchers (Dr S. Music, Dr M. Ristic and Dr S. Krehula) from Department of Chemistry of Materials, Rudjer Boskovic Institute, Zagreb, Croatia as well as Dr B. Dojcinovic from Institute of Chemistry, technology and metallurgy (Belgrade, Serbia), J. Pantic (INSV) and D. Popovic, from FEI were invited lecturers.

The lectures were devoted to the advanced synthesis routes and application of different experimental techniques that are useful in the characterization of nanostructured materials with the focus on the investigation of materials properties that are crucial for the development of biomedical applications. Trainings were focused on:

• The synthesis methods for obtaining doped iron oxide nanoparticles and application of spectroscopy methods,
• FTIR (Fourier Transform Infrared) and Mossbauer Spectroscopy (Prof S. Music, Dr S. Krehula) in materials characterization,
• Surface and colloid chemistry of iron oxides, hydrothermal and solvothermal synthesis (Dr M. Ristic),
• Scanning Electron microscopy investigation and methods for micrographs analysis (Dr M. Ristic).
• Introduction to Transmission Electron Microscopy and state of art devices (D. Popovic),
• Potential applications and results obtained by implementation of ICP (Induced Coupled Plasma) technique (Dr B. Dojcinovic),
• Application of optical methods in the investigation of nanostructure ceramics (Dr J. Pantic). Guest lecturers presented used procedures for sample preparation and details of structural characterization, including experimental setups, data analysis as well as its interpretation.

Held in the first project year, this Workshop gave the introduction to different methods for iron oxide NPs preparation and made a solid basis for the application of experimental techniques, which are not commonly used in the characterization of nanomaterials in our Laboratories.

B.2) Workshop on Advanced techniques for physico-chemical characterization of MNPs was organized with Dr Beatriz Sanz Sagué, from Zaragoza, Spain, an expert in magnetic hyperthermia measurements that participated in construction and measurements on DM device. In newly established department for magnetic hyperthermia, Dr Beatriz Sanz explained the aspects of magnetic hyperthermia and demonstrated measurement procedures to MagBioVin team members, students and collaborators from the “Vinca” Institute and University of Belgrade. The experimental work was carried out in groups with 3-5 participants during 6 days of trainings. Researchers have gained the experience in:

• Basic principles of magnetic hyperthermia,
• Optimization of thermal heat generation ability,
• Optimization of thermal homogeneity in the target tumour,
• Limit heating zone region close to the tumour without damaging the healthy tissue,
• The relationship between magnetic properties of MNPS and SAR,
• Sample preparation for magnetic hyperthermia and Clinical application of magnetic hyperthermia.
• Measurements with the hyperthermia device,
• application of Software MANIAC,
• SAR measurements of suspensions of MNPS and
• Calculation and interpretation of SAR values.

Adopted skills have become a daily routine in further scientific work of MagBioVin team members.

B.3) Workshop "DNA damage induced by radionuclide labelled functionalized MNP" was organized with a help of Prof Germana Meroni from Molecular Genetics lab, Department of Life Sciences, University of Trieste, Italy, who gave the talk about TRIM E3 ubiquitin ligases in health and disease and opened wide discussion about the state of the art research topics from the field of bioinformatics tools application in genetic research. Practical part of the Workshop was led by Danilo Licastro from Cluster in Biomedicine, AREA Science Park - Basovizza campus, Trieste. It was devoted to Bioinformatics Pipeline.

Ten MagBioVin researchers had an opportunity to perform analysis of gene expression data originating from our previous experiments and public available data sets. Differentially expressed genes were mapped onto molecular pathways, gene regulatory networks, and different subsets were characterized with biological meaning. Upstream analysis was performed for the identification of potential regulators of specific gene expression patterns. The analysis required the employment of tools necessary for the preparation and analysis of large gene sets: Gene Set Enrichment Analysis – GSEA, R/Bioconductor, ROAST: rotation gene set tests for complex microarray experiments, Database for Annotation, Visualization and Integrated Discovery –DAVID.

As the understanding of computational algorithms for bioinformatics analysis of large sets of differentially expressed genes makes an irreplaceable method for extracting of biological meaning from the gene sets and for planning of validation experiments, the lecture was of great benefit for MagBioVin researchers.

B.4) Workshop "Intellectual property, innovation promotion and technology transfer", held for MagBioVin team members, representatives of stakeholders, broader scientific public as well as representatives of Republic of Serbia Intellectual Property Office. The main goals of the Workshops were to clearly define pathways from idea to licence or spin-out technology and to train team members in the field of Intellectual Property Rights, its importance and proper procedures used in the innovation protection. The lectures covered:

• Fundamentals of intellectual property,
• Foundations of intellectual property rights,
• Evaluation of technology as well as licensing agreements to the intellectual property rights,
• Clauses on confidentiality and ways of their formulation,
• Operative procedures for spin-out units, and
• Various phases of production and the creation of prototype.

All the topics were followed by examples from the practice. The lecturers were Tamara Colic Milosavljevic, transfer technology manager from the Center for Technology Transfer, University of Belgrade on a topic ‘From idea to licence’, Era Chair Dr Zeljko Prijovic, an eminent expert in biochemistry and patent holder, with the talk about ‘Patent as a tool for innovation protection in science: benefits and pitfalls’, experts of the Education and Information Center of the Intellectual Property Office, Republic of Serbia Daniela Zlatic Sutic, Head of the Center, Natasa Milovanovic, Patent Examiner, EIC member, Department for mechanical and electrical engineering and Ivana Krdzic, BSc, Patent Examiner, EIC member, Department for chemistry and chemical technology who held the lectures on intellectual property with the focus on the Intellectual Property rights. The introduction lectures were devoted to the presentation of Patent protection system as a tool to protect an invention which is new, involves an inventive step and is susceptible of industrial application. Afterwards, the detailed representation of the patent system was organized to demonstrate the principles of national and international patent protection.

B.5) The workshop entitled ‘XRPD line broadening analysis and microstructure parameters’ was a three-day long course which took place at the Vinca Institute, in the Laboratory for Theoretical and Condensed Matter Physics. The subject of the course corresponded to one of the highest demanded tool for researchers involved in magnetic nanoparticle synthesis. In modern research, progress in both instrumentation and data analysis facilitates systematic size–strain crystallographic studies, which together with magnetic measurements provide better understanding of relationships between microstructure and magnetism. The educational level of the workshop participants was heterogeneous: PhD students, post-doc researchers, senior researchers. The educational profile was divergent as well, including technologists, chemists and physicists.

The aim was to improve the skills of those MagBioVin scientists already engaged with high-level X-ray diffraction technique and crystal structure characterization, but also to serve as an upgrading course for those who were more at basic or intermediate level in that field. An eminent Serbian crystallographer, Prof. Dr Aleksandar Kremenovic was the invited lecturer for this workshop. The seminar was divided into three parts, each reserved for the duration of one day. Topics of this workshop were:

• Width of a diffraction line and factors that are responsible for XRPD line broadening,
• X-ray line broadening dependence on microstrain and crystallite dimension,
• Separation of instrumental from size-strain XRPD line broadening,
• Methods for determination of microstructure parameters,
• Accuracy and limitation of XRPD line broadening.
• XRPD line broadening analysis and microstructure parameters – the use of computer program FullProf,
• XRPD line broadening analysis and microstructure parameters – the use of computer program Breadth.

The final day of the workshop was dedicated to practical interactive work on previously collected XRPD data. Attendees were divided in 5 groups of 3 researchers, and were given two microstructure problems to solve by using programs FullProf and Breadth, under Prof. Kremenovic supervision. The most important outcome of this workshop was provision of significant knowledge upgrade for the MagBioVin team members in the highly specialized field.

C) Lectures and trainings given by invited experts

During the project implementation, ERA Chair holder has invited several eminent experts in fast developing fields of nanomedicine to present the latest trends and the latest knowledge in the topics of interests. It should be emphasised that outcome of these trainings were introduction of the above mentioned methodologies as routine protocols in the research of the MagBioVin team. In addition, two experts for project management were invited as well, to transfer the knowledge regarding both organizational and financial aspects of EU funded projects. These visits are shortly described below.

C.1) During the visit of Professor Ian Bruce from Nanobiotechnology Research Group, University of Kent, MagBioVin team members had opportunity to learn and discuss about multidisciplinary research in the field of molecular biology, organic and inorganic chemistry and materials science. The eminent expert from the field of Nanobiotechnology gave lecture with the principal themes focused on structured nano- and micro-composite materials and Surface activation and modification chemistries.

C.2) Dr José Mariano, Assistant Professor from the Department of Physics, Faculty of Sciences and Technology, University of the Algarve, Portugal worked with the MagBioVin team members from the Department of Theoretical and Condensed Matter Physics (DTCMP): Bratislav Antic, Vladan Kusigerski, Marija Perovic and Jovan Blanusa, and PhD students Marko Boskovic and Milos Ognjanović. During his several months long stay, his activities were focused on the application of hyperfine interactions spectroscopies, namely Mossbauer spectroscopy and Ferromagnetic Nuclear Resonance (FNR), to the study of magnetic nanoparticles relevant for hyperthermia applications. Dr. José Mariano held lab sessions in order to demonstrate to “Vinca” staff and students the advantages of using these techniques in the analysis og MNPs typically in use in the frame of the MagBioVin project. He provided a hands-on introductory course on rapid-prototyping techniques for the development of digital and micro-controller electronics, including firmware and software, to be applied to the development of scientific instrumentation, namely the Specific Absorption Rate (SAR) equipment being developed at “Vinca”. Dr. José Mariano held lab sessions to introduce the subject to the students. These lectures were followed by actual development work on the hardware and software of the SAR measuring equipment. The course was organized in four modules: Introduction to digital electronics and microcontrollers; Development of microcontroller applications using open-source platforms; Development of microcontroller applications and digital electronics using FPGA; Hands-on development of a digital instrumentation module.

C.3) Dr Renata Mikolajczak, Assistant director for Science, National and international cooperation from National Centre for Nuclear Research, POLATOM Center, Otwock, Poland was the guest of the Institute "Vinča" . During her career, Dr Mikolajczak has been studying the conditions and possibilities of marking different ligands and nanoparticles with different radionuclides: beta emitters (Y-90, Re-188, Lu-177, I-131 ...) for therapy, gamma emitters (Tc-99m, ..) for diagnosis and monitoring of therapy, and In-111 for therapy planning and dosimetry. Today, POLATOM is one of the leading centres for the production of reactor radionuclides, as well as radiopharmaceuticals that are applied in nuclear medicine centres in Europe and around the world. The use of radionuclide labelled magnetic nanoparticles in synergy with magnetic hyperthermia in cancer combat is one of the main research topics of the MagBioVin team. Thus the visit of Dr Mikolajczak as one of the peer experts in the production and use of various radionuclides was of the immense importance in knowledge transfer in respect to this research field. She had transferred her rich experience to MagBioVin researchers during the round tables and discussions, as well as during the practical demonstrations in the facilities of the Laboratory for Radioisotopes.

C.4) Marija Mali, an eminent financial expert in International Project Management from ‘Jozef Stefan’ Institute, Ljubljana visited the "Vinca" Institute with aim to transfer and improve knowledge about project management from financial and legal standpoints. During the discussion with MagBioVin Management Board Members, the topics concerning H2020 Projects Regulations were elaborated. She also demonstrated the preparation and updating of the documents required by the European Commission for the project reporting. The guest had several working meetings: with the head of the "Vinca" financial department (Marija Đinović); with the Institute employees responsible for financial aspects of national projects, and with the Head of International Projects Office (Dr Snežana Pašalić). The main goal of these meetings was to improve the existing system of projects' financial management, to initiate changes in current procedures in order to harmonize them with the practice in EU countries, and to increase the management efficiency regarding the number and quality of applications for international funding.

C.5) Within this topic, we can also mention a visit of Dr Tapas Sen, from School of Physical Sciences and Computing, University of Central Lancashire, Great Britain, visited Vinca Institute. Dr Tapas was invited due to his large experience in managing Euro projects (worked on multimillion Euro projects under EU framework V and VI programmes) and multinational projects in collaboration with industry. At the same time, his area of research interest fits tightly with MagBioVin project’s research topic. During his stay, several discussions regarding project management as well as future scientific course were held.

D) Accreditation of the new Center of Excellence for Nanomaterials Research and Development

MagBioVin support in reinforcement of human and material resources has made a solid ground for the establishment of the new Center of Excellence in the "Vinca" Institute. The core personnel of this Center consist of MagBioVin team members under the ERA Chair supervision. The main field of the Center’s expertise is designated to be R&D of new nanomaterials for various applications, with the primary focus on biomedical applications. Founded under the "VINCENT" acronym, the Center consists of six subunits (departments):

1. Department for nanomaterial synthesis
2. Department for structural and microstructural analysis
3. Departmemt for magnetism and theoretical modeling
4. Department for magnetic hyperthermy and biosensors
5. Department for radiolabeling and bioanalytics
6. Department for tissue culture, biochemistry and molecular biology

An integral part of the Centre (located within the 6th department) is animal house for immune-compromised animals with capacity of over 500 animals ("Vivarium"). By its establishment the "Vinca" Institute obtained the prominent upgrade and a facility that is unique in Serbia. This facility satisfies requirements for breeding, housing and experiments in vivo. The animal studies confirmed to ethical guidelines and complied with the European Council Directive (86/609/EEC) and the rules for animal care proposed by the Serbian Laboratory Animal Science Association (SLASA).

The official procedure for accreditation of the Center of Excellence was initiated at the Accreditation Board of the Serbian Ministry of Sciences and Technology. All the steps of this procedure and necessary documentation that must be submitted are prescribed by the applicable laws. Evaluation of the proposal as well as issuing of the accreditation license is done by the aforementioned Accreditation Board. The proposal has been successfully finished and submitted in the previous project period. In the last reporting period the remained steps of the procedure for the accreditation of the Center of Excellence has been successfully completed, and so the VINCENT Center has been registered by the Serbian Ministry of Science as the new constitutive unit of the “Vinca” Institute of Nuclear Science.

It should be emphasized as well that this appointment represented a valuable contribution to the efforts of official accreditation of the "Vinca" Institute as an institute of a national interest, which was one of the Institute’s strategic plans.

3. Reinforcement of experimental infrastructure

Regarding the reinforcement of equipment recourses, the MagBioVin project has provided an exceptional support to the "Vinca" Institute by upgrading its abilities related to hyperthermia and cancer research up to the state-of-the-art level. Equipment procurement was performed in two stages: the first one was foreseen by the initial GA, and was completed during 2015, while the second was done in accordance with amendments to the GA approved by EC during 2018.

The first stage of equipment provision resulted in installation of two crucially important devices:

1) Rigaku Smartlab diffractometer with small angle scattering unit (SAXS), and
2) Hyperthermia measurement device (DM model by "Nanoscale Biomagnetics").

The Smartlab diffractometer is specially configured to allow for a fast and reliable nanoparticle structure and microstructure analysis, which is essential during the stage of nanoparticle synthesis. However, what makes the qualitative step forward with this device is its ability to perform small angle scattering measurements. The "Vinca" Institute did not (and still does not) have a separate unit specialized for SAXS technique at its disposal, so the SAXS option added to the Smartlab diffractometer has proved to be a significant improvement in acquiring this characterization method. On the other hand, the DM hyperthermia analyzing device is a unique kind of equipment aimed specifically to explore hyperthermia effects in vitro, in cell cultures and in vivo on small animal models, all in one assembly.

Possessing advanced technology device like DM hyperthermia analyzer opened additional possibilities, which go beyond measuring application; MagBioVin team was engaged in the "RADIOMAG" COST action, participating in the standardization of protocols for SAR (Specific Absorption Rate) measurements at European level.

Both devices set the new level in research process and quality, and fully confirmed the equipment related assessments formulated in the project GA. However, as research activity level raised during the years following the purchase, few of weak points became noticeable as well, especially concerning availability of imaging techniques for tracking nanoparticle and drug distribution in living organisms. In some other cases, due to significant increase of workload, the insufficiency and shortage of existing capacities have become evident.

Accordingly, the procedure for necessary amendments to the GA has been initiated, and on 1stOct.2018 European Commission has officially approved the amendment N° 3 to the grant agreement of MagBioVin project. On the basis of accepted changes within Annex I "Description of Work", the provision of additional pieces of equipment was done:

1) Small lab equipment;
2) Electrochemical workstation Autolab;
3) Multimodal in vivo imager;
4) Software for Rigaku diffractometer;
5) Fluorescent microscope;
6) Biosafety cabinet;
7) Cryotome.

Facilities for the synthesis were significantly upgraded by the provision of the furnace and specialized autoclave for high pressure polyol-hidrothermal synthesis, and the vacuum furnace custom-made to provide both high temperature synthesis and measurement ability in anaerobic and suppressive conditions. These devices are purchased under (1), as "small lab equipment". This category includes items that served primarily to fill (minor) gaps in research process, by making the "smaller steps" available on site.

Some of the above mentioned equipment (enumerated under 1, 4, and 6), served to expand existing capacities to the level that sufficiently responds to increased intensity of research activities and number of involved personnel. Others (items under (2), (3), (5) and (7)) represent introduction of a new quality into research process, by providing access to technologies that were unavailable to MagbioVin team either at the Institute or national level.

This especially holds for the multimodal in vivo imager, which overrides increasingly difficult access to scanning techniques for small animal models. In vivo Bioluminescence imager (BLI imager) is an innovative device that allows monitoring of tumor therapy effects and other types of biological testing in experimental animals in vivo, without sacrificing animals. It represents a fresh alternative to costly animal MRI devices, and it is therefore perceived as essential instrument in future investigations and bio-applications of hyperthermia or any other research comprising live animal models. It was also the subject of the largest approved budget for a single purchase within MagBioVin project (200.000 EUR).

Smartlab diffractometer, DM hyperthermia measuring device and BLI imager, together make the framework of the future "Vinca" Institute research capacities that are unique for both Serbia and Balkan region as well.

4. Reinforcement of international cooperation - Joint project proposals

The realization of MagbioVin project gave a particular boost to the establishment of new connections between scientists from the "Vinca" Institute and researchers abroad. Starting from the middle of 2016., members of the MagBioVin team were engaged in writing 22 proposals for international projects calls in overall. Compared to the previous rate of 2-3 proposals per year from laboratories participating in MagBioVin project, an increment of almost 5 times has been was achieved.

Of these 22 proposals 3 were aimed for Horizon 2020 call, 3 for regional (Danube region) calls, one for IAEA (International Atomic Energy Agency) call and 14 for bilateral collaborations between Serbia and other countries (of predominantly European origin). Although there is an obvious space for further improvement of the success rate, it should be emphasized that MagbioVin team has obtained funding for one EUREKA project (funds: 150,000 EUR), 10 bilateral cooperation projects (overall funds of about 50,000 EUR), and has one IAEA proposal that is still under evaluation at the time of writing this report (funds: 194,000 EUR). The large number of obtained bilateral cooperation projects is of particular significance despite appreciably modest funding. In fact, it represents an important opportunity for supporting of newly established contacts, and a framework for future larger scientific collaboration and common project proposals.

II. Improvement of quality and expansion of research fields (S&T results)

The support for human and material recourses by MagBioVin project resulted in progress made in research quality, and that consequently led to considerable increase in number and impact of published scientific results as well. The development of research spectrum was in fact much wider than application of hyperthermia and targeted radiotherapy. The most important S&T achievements can be classified in several different fields:

1) Magnetic hyperthermia

Using the novel equipment setup, MagBioVin researchers have conducted the systematic study of specific loss power (SLP) of polydisperse water based ferrofluids, Fe3O4/PEG200 and Fe3O4/PEG6000. Specific loss power was measured in different fields at fixed frequency of 580.5 kHz. Though labeled as superparamagnetic by magnetization measurements, SLP field dependence showed deviation from the behavior predicted by commonly employed linear response theory. The scope of this theory, as well as of Stoner-Wohlfarth based theories, for both samples with wide size distribution was discussed. Deviation from the expected behavior was explained by referring to polydisperse nature of the samples and different heating mechanisms that can coexist in such system.

2) Synthesis, coating and radio labelling of magnetic nanoparticles

Members of the MagBioVin team have succeeded in preparation of 99mTc–bisphosphonate–coated magnetic nanoparticles (MNPs) based on Fe3O4. These particles have sufficiently high in vivo stability and heating ability for possible application in biomedical fields. The presence of the bisphosphonates on the MNPs surface, enabled their biocompatibility, colloidal stability and successful binding of the radionuclide. Coated MNPs were radiolabelled with 99mTc using stannous chloride as the reducing agent in a reproducible high yield (95 % for Fe3O4–MDP and 97 % for Fe3O4–HEDP MNPs) remaining stable in saline and human serum for 24 h.

3) Biodistribution studies of magnetic nanoparticles

The biodistribution studies presented significant liver and spleen uptake in healthy Wistar rats after intravenous administration at all examined time points, due to the colloidal nature of 99mTc–MNPs. Results of gamma scintigraphy are in accordance with biodistribution studies demonstrating high in vivo stability of radiolabelled MNPs and therefore results of both methods were proved as accurate in vivo information on the biodistribution profile of investigated MNPs.

4) Electrochemical sensing

A disposable acetaminophen biosensor based on inkjet-printed CNT electrodes (IJPCNT) modified with amidase/cerium dioxide@graphene nanoribbons composite was developed (ACeO2@GNR/IJPCNT). The enzyme amidase A was used for the first time as a recognition element. Inkjet-printed CNT electrodes served as a basis for the construction of a biosensor that enables droplet detection using 5 μL sample volume. The biosensor showed high selectivity, sensitivity and low detection limit. This approach allows fast and reliable detection of acetaminophen in biological fluids with negligible matrix effect and remarkable reproducibility.

5) Nanomaterials for catalysis - Electrochemical approach

MagBioVin team has succeeded to develop a novel platform for the removal of the synthetic organic dye Reactive blue 52 based on a screen-printed electrode (SPCE). Additionally, SPCE was supported on a nanocomposite obtained by decoration of reduced graphene oxide (RGO) with iron oxide nanoflowers (IONFs), labelled as IONF@RGO/SPCE. IONFs were synthesized by polyol mediated reduction of iron (III) chloride and characterized. IONF@RGO nanocomposite was prepared using a microwave hydrothermal assisted procedure. The high stability (service life) of the IONF@RGO/SPCE electrode was measured, and it remained almost unchanged over time, achieving the same removal efficiency after 50 cycles of usage. Impedance tests (EIS) indicated the synergetic effect of the used materials (two-component composite) by reducing resistivity of the system and improving its catalytic activity, which was confirmed with cyclic voltammetry (CV tests) where the great increase of the electrochemically active surface area sites was obvious.

6) Nanomaterials for adsorption of toxic metals

To the best of our knowledge, the MagbioVin team was the first one to report the impact of substitution of iron by yttrium ions on the morphology of magnetite-based nanoparticles. While Fe3O4 particles were mostly pseudospherical with relatively uniform size distribution, partial substitution of iron by yttrium leads to inhomogeneity in morphology: appearing nanorods and broadening distribution of pseudospherical particles. With increasing concentrations of yttrium, the quantity of nanorods was increased, and aggregates of pseudospherical nanoparticles were formed. Importance of this subject for future designs of magnetite-based sorbents has found its ground in the role of morphology/yttrium substitution in inorganic arsenic (III) and arsenic (V) removal. Improved adsorption performance of Fe3-xYxO4 nanoparticles achieved by increasing of Y concentration is very important result for the technology of purifying underground and wastewater, and can be important guideline for future research in the area of design magnetite-based nanoparticles for waste water treatments and many other technologies.

7) Biosensing

MagBioVin researchers have proposed a new composite material with enhanced electro catalytic properties as a novel screen-printed sensor for the quantification of NADH. Additionally, the surface was modified with alcohol dehydrogenase for the preparation of amperometric biosensor for analysis of ethanol. Under optimal conditions, the working linear range and limit of detection for ethanol sensing was 1–1800 µM and 0.19 µM, respectively. For NADH, the linear range was from 1 to 1300 µM with limit of detection of 0.52 µM. Moreover, effects of some possible interfering compounds were investigated and the developed procedure was applied to commercial alcoholic beverages. The results obtained showed satisfactory precision and accuracy of the developed method and confirm the proposed approach could be a possible replacement for the currently used techniques for ethanol and NADH quantification.

8) Toxicity study of DE-EDCP

One encouraging discovery made by MagBioVin team was that novel OO'-diethyl-(S,S)-ethylenediamine-N,N’-di-2-(3-cyclohexyl) propanoate dihydrochloride (DE-EDCP) displayed in vitro antiproliferative activity on several human and mouse cancer cell lines. In order to reveal its toxicity profile, acute and repeated-dose toxicity studies were performed in Naval Medical Research Institute (NMRI) on Han mice. In the subacute toxicity study, DE-EDCP was administered intravenously. There was a significant increase in urea and alanine aminotransferase in female mice and aspartate aminotransferase and alkaline phosphatase in both genders. The histopathological changes confined to the liver and kidney, but in other organs were not found. Satellite group revealed that changes in the kidney and liver were less pronounced, suggesting their reversibility. Interactions with DNA could also be of importance for understanding DE-EDCP toxic side effects.

9) Design and construction of Dynamic Hysteresis Magnetometer for SAR measurements

Development of a standardized method for experimental determination of SAR (Specific Adsorption Rate) values is among the priorities of many research teams worldwide involved in magnetic hyperthermia investigations. Until recently the typical method for SAR measurement was based on calorimetry, however several of its shortcomings initiated the quest for a method that is more straightforward in terms of construction of an experimental device and reproducibility of the measured data.
Recently a new approach appeared which is based on a spin dynamics in alternating (AC) magnetic fields as an underlying physical phenomenon responsible for the appearance of magnetic heating. The principal relays on the hysteretic behavior of magnetic spins exposed to AC magnetic fields and the fact that he area of the dynamic magnetization (hysteresis) curve is proportional to SAR. Since this method is considered in a research community as a promising upgrade of a calorimetric method, MagBioVin team members started with the “in house” design and construction of a dynamic hysteresis magnetometer. So far the construction of the device has been finished and first stage testing and calibration successfully completed. In the second stage, certain improvement in construction is foreseen as well as development of the software package for control, acquisition and processing of the data. It should be emphasized that participation of MagBioVin members in the COST action RADIOMAG (which was devoted to standardization of equipments and protocols for hyperthermia investigation) was of crucial importance since it enabled contacts with peer experts in the field for exchange of ideas, knowledge and experiences.

Potential Impact:
A. The potential impact of the project
The MagBioVin project is coordinated by the "Vinca" Institute (Belgrade, Serbia), the largest multidisciplinary R&D institute in Serbia and among the largest institutions of this kind in the West Balkans (WB) region with considerable international reputation. Consequently, successful realization of the project which implies improved networking, visibility and integration of "Vinca" at international level is expected to have a positive impact on both regional level as well as on the European Research Area (ERA) as a whole.

A.1. Strengthening of the human and experimental resources, research excellence and good practice in the "Vinca" Institute
During its seventy years long tradition “Vinca” successfully cultivated multidisciplinarity of its researchers, and so nowadays Institute consists of 13 laboratories, 7 research centers and more than 300 PhD staff working in different areas of science. The MagBioVin project unites experienced researchers with diverse scientific backgrounds to work in synergy on the common program. This will ensure necessary coordination of knowledge and presence of critical mass of experts for complex multidisciplinary research. In this way MaгBioVin project will remarkably raise the level of cooperation in the “Vinca” Institute to a much higher level than before which in addition presents a solid basis for sustainability of multidisciplinary researches in the future.

Prospect of the political and economic integration of the regions of convergence and WB in European Union brings into focus the increase of research and development quality as close as possible to high European standards. Therefore among the principal objectives of the MagBioVin project is to qualify the MagBioVin human resources and material facility for the work in the highly specialized area of biomedical applications of magnetic nanoparticles (MNPs) which addresses at least two priority activities (Nanosciences and Health) within the Cooperation Programme of the FP7 initiative.

In respect to human resources, it should be emphasized that selection of a prominent scientist for ERA Chair holder and subsequent recruitment of recognized scientists under his supervision considerably reinforced the capabilities of the MagBioVin research staff. Newly recruited researchers (two experienced scientists and three post docs) brought new knowledge and skills that were previously underdeveloped and substantially raise the research potential of the team. Moreover, dissemination of new knowledge and know-how among the "Vinca" permanent staff during the project lifetime ensures the sustainability of the attained high knowledge level after the end of the project. We would like to stress that four of the researchers hired for the project obtained positions in "Vinca" (ERA Chair holder, two post docs, and one PhD student) after the end of the MagBioVin project, as well as Project Manager. In this way, project significantly contributed to a brain-gain efforts in the Institute.

Acquisition, development and upgrade of MagBioVin research equipment has been successfully done, and the new equipment was incorporated in the existing laboratories which increased the overall research capacities of the Institute. It is expected that the impact of the laboratory equipment upgrade will add value not only to a research and development potential of the Institute, but also will enable training of young researchers and PhD students in exploiting state-of-the art equipment. Additionally, new equipment will also be used for joint research activities with regional and EU Networking partners, both from academia and industry.

Introduction of the new and widening of the existing research topics is a result of the reinforced human and experimental potentials of the "Vinca" Institute. The list of the most important topics is given below:
- New methods for the synthesis of encapsulated (coated) MNPs and their functionalization;
- State-of-the-art nanocrystallography methods for analysis of nanoparticle microstructure:
WPPM (Whole Powder Pattern Modeling) and PDF (Atomic Pair Distribution Function);
- New theoretical methods for: (i) modeling of both magnetic hyperthermia and radiation
therapies; (ii) MRI image analysis;
- New methods of colloid chemistry for obtaining ferrofluids suitable for MRI;
- Manufacturing of radionuclide labelled MNPs;
- In vitro/in vivo (animal model) manipulation and monitoring of labelled MNPs;
- Implementation of magnetic hyperthermia device for in vitro/in vivo experiments;
- Development of techniques for monitoring of the therapeutic effects on the cancer cells in
vitro/in vivo (animal model).

Accreditation of the new Center of Excellence “VINCENT”- Reinforcement of both human and materials resources of the ‘Vinca’ Institute achieved through the MagBioVin project has made a solid ground for the establishment of the new Center of Excellence in the Institute. The core personnel of this Center consists of MagBioVin team members under the ERA Chair supervision. The main field of the Center’s expertise is designated to be R&D of new nanomaterials for various applications, with the initial focus on radiolabeled nanomagnetic materials for biomedical applications. The procedure for the accreditation of the Center of Excellence has been successfully completed, and so the VINCENT Center has been recognized by the Serbian Ministry of Science as the new constitutive unit of the Vinca Institute of Nucleas Science. It should be stressed that this appointment represented a valuable contribution to the efforts of recognizing the Vinca Institute as an institute of a national interest, being one of the Institute’s strategic plans.

Improvement of research management was achieved by gaining new experience and knowledge brought by ERA Chair holder as well as through communications with peer scientists from Strategic partners labs and others from ERA during project execution. Activities like hiring of new staff, acquisition of equipment and broadening of research topics certainly required improvement of research management due to a elevated complexity of the research process. Increase of manpower enabled project leaders to invest more effort in scientific management activities and give them more potential to cope with research topics, thereby increasing competitiveness of the Institute in the environment of ERA. Also, the improved management could bring closer the advanced research topics to the needs of industry (in both Serbia and EU), and in that way strengthen ties between research institutions and industrial companies. This will result in two-way benefit, research in Serbia will be improved through better financing and the industry will benefit through links with the state of the art knowledge, methods and practice. All of this will intensify the connectivity of regional industry and research institutions with European enterprises and standards valid there.

Prevention of the “brain-drain” process that had severely affected the human potential of Serbia during the last twenty years. Increase of the research potentials up to high standards of EU research centers substantially raise of the working conditions and thus support the motivation of talented scientists to develop their careers in the country. Both mobility of researchers and visits of the key experts from abroad fostered not only the increase of the overall knowledge in the relevant field but also developed cooperative approach relying on the cross-cultural exchanges. In addition, by recruitment of respectable Serbian scientists having left the country the "Vinca" Institute even partially reversed the brain-drain process. It should be emphasized that the ERA Chair holder (Dr Zeljko Prijovic), peer Serbian researcher who returned from the abroad, obtained permanent position in "Vinca" after the end of the project.

A.2. Improved networking, visibility and competitiveness of the "Vinca" Institute at regional and European level
One of the project objectives was to achieve better integration and visibility of the "Vinca" Institute by improving the MagBioVin team networking on regional and European level. It should be point out that team members had already developed substantial collaboration with many institutions abroad, but majority of them relied on bilateral projects while larger scale integration was scarce.

In the first stage, reinforcement of collaboration has been achieved with the project Strategic Partners: Nanoscience Institute of Aragon, Zaragoza (Spain), Institute of Mathematics, Physics and Mechanics, Ljubljana (Slovenia), The East Paris Institute of Chemistry and Materials Science (EPICaMS - ICMPE), Paris (France), and Institute of Experimental Physics, SASK, Kosice (Slovakia). These are research institutions from EU that committed to provide help in project execution by delegating their peer scientist to Steering Committee as well as lecturers for workshops in "Vinca". Additionally, they expressed goodwill to host project team members for training purposes. Through these activities exchange of people, knowledge and ideas has been achieved, which reinforce the collaboration with partner institutions. At the same time, team members were involved in some of the collaborative work within the existing EU research networks of the partners thus expanding the existing contacts and cooperation.

Participation of team members at scientific meetings of high level (conferences, seminar, workshops) were used not only for dissemination purposes but also for establishing a new contact with scientist from the eminent research institutions from the abroad. These opportunities were used to discuss possibilities for collaborative work and to make a preliminary schedule for mutual visits in case of expressed interest.

Participation of team members in COST actions was utilized as another resource for expanding contacts within the scientific community from the similar research area. In the project period MagBioVin team members participated in two COST action, TO-BE (MP 1308, 2014-2018) and RADIOMAG (TD 1402, 2014-2018). Active participation in these actions as members of working groups, co-authors of publications issued in a course of these actions, and involvement of young researchers in STSMs (Short Term Scientific Missions) improved visibility of the project team, established new contacts, and increased awareness on the abilities of the project team among wider scientific community.

By utilizing the reinforced collaboration with partner institutions and newly established contacts MagBioVin team participated in making and submission of more than twenty project proposals for different funding schemes: one national project, bilateral projects (with France, Slovenia, Croatia, Slovakia, and Montenegro), regional projects (Danube initiative), one IAEA project (under review), ЕUREKA project and H2020 projects. It should be emphasized that funding was obtained for 11 projects, 10 bilateral and one Danube initiative, with the total budget of almost 500.000 EUR.
The increasing excellence, as a result of the above mentioned running projects, together with the outcomes of the MagBioVin project will lead to the prominent position of the “Vinca” Institute in the region. From such a position it will act as clustering centre for future regional networking which will ensure the high impact on the regional supporting scheme. As a consequence, this will increase the Institute visibility and its impact on the wider region, with ERA as the prime area of interest.

The "Vinca" Institute as WB leader in radiopharmaceutical production, with the focus on both diagnostic and therapy products. Due to the longstanding experience and accumulated knowledge of its scientists and increased excellence as a result of the MagBioVin project, the "Vinca" Institute complies with all conditions necessary for work with ionization radiation. "Vinca" has a unique platform in WB region consisting of laboratories for the research, development, production and distribution of radiopharmaceuticals for diagnostic and therapy. Consequently, a continuous access to radiopharmaceuticals enables interdisciplinary and cooperative investigation of new diagnostic and therapeutic agents that are in deficit at radiopharmaceutical market. Although tumor therapy based on nanostructured materials can be very effective in some cases it is still insufficiently used because of the novelty of the technique and the difficulty in the production and distribution of a therapeutic radiopharmaceutical. The involvement of small R&D companies and pharmaceutical companies in MagBioVin project is essential since the whole palette of the agents for tumor therapy is planned to reach the market.

B. The main dissemination activities and exploitation of results

B.1. Dissemination activities are planned to act in synergy with other main objectives of the MagBioVin project.
Dissemination to scientific community - Reinforcement of the “Vinca” Institute experimental facility and its human resources will result in the raise of the research quality and thus enable the deeper involvement into the cutting-edge research topic such as biomedical applications of magnetic nanoparticles (MNP). Consequently, the scientific outcome of such research is publishable in peer-reviewed international journals and scientific meetings (conferences, workshops, etc.) of high level, mainly covering the following topics: physics and chemistry of nanomaterials, radiation physics and chemistry, biosensors, biochemistry and biomedicine. During the project lifetime team members published several tens of papers and presented their results at major international conferences devoted to research topics of interest.

Dissemination to Universities - Important target group for dissemination of knowledge comprehend young researchers from the field doing their PhD or post-doctoral studies. MagBioVin team members in cooperation with the universities in the country participate in organizing training courses and demonstrations for young researchers at the contemporary facilities of the “Vinca” Institute. During these activities young researchers are getting deeper insight into high-level scientific activities at the early stage of their education and training which will raise the overall level of their abilities and skills, and enable their easier involvement into the cutting-edge researches at the later stages of their careers.

Dissemination to stakeholders from medicine/pharmacy - In order to achieve impact on the national level special attention is paid to institutions involved in biomedical investigation (medical centers and institutes, R&D departments of pharmaceutical companies, etc.), since they represent the group that is most promising for the exploiting of the project results. Several joint meetings have been initiated and organized with their representatives in order to promote the main project ideas and its outcomes. These were also utilized as a good opportunity to get the feedback from the key stakeholders in order to increase the level of understanding of their needs. It should be emphasized that in spite the economic crises and isolation during 1990’s medicine and pharmacy remains among the most vital and prospective areas in Serbia with substantial investments during the last decade. The longer term outcome of the MagBioVin is the application of the research results in production and use of biocompatible MNP-based materials in diagnostic and therapeutic practice.

Dissemination to policy makers - The particular effort is aimed towards spreading of project results to decision making structures in the country, including relevant Ministries (Science, Health) and industrial enterprises (pharmacy and medicine). This is an important prerequisite in terms of influencing further budget decisions of certain targeted groups and potential project stakeholders. For instance, multidisciplinary research aimed towards biomedical applications will certainly deserve attention of the Ministry of Health and it will influence its future budget planning for the support of such investigations. Furthermore, the majority of MNPs assignments are aimed towards cancer combat which has one of the highest priorities in the health strategy of Serbia due to the steep increase in number of deceased from malignant diseases in the last decade. Thus the MagBioVin results are expected to be exploited within the framework of a wide national initiative towards cancer combat by supporting interdisciplinary investigation aimed to development of new diagnostic and therapeutic agents. The projected outcome of this initiative is substantial improvement of diagnostic and treatment strategies for patients with widespread diseases.
Besides contacts with the stakeholders and policy makers at the national level members of the MagBioVin team were also active at the international stage by participating in the work of several organizations and bodies with the important role in shaping the policies regarding application of nanomaterials and radiopharmaceuticals in medicine. Two scientists from project team were members of the Serbian official delegation at the 62nd Annual Regular Session of the IAEA General Conference held from 17th to 21st September 2018 at the Vienna International Centre (VIC) in Vienna, Austria. MagBioVin team members also actively participated in the work of a Committee for a new ISO standard on the manufacturing of radiolabelled nanomaterial, during 12th International Conference on the Scientific and Clinical Applications of Magnetic Carriers, Copenhagen, Denmark, from 22nd to 26th May 2018. During the 8th Balkan Congress of Nuclear Medicine (BNMC 2019) in Bodrum (Turkey) the Head of the Laboratory for radioisotopes (Dr Sanja Vranjes-Djuric) and member of the project MB had several discussions and meetings with the leaders from the main Centres for radiopharmaceutical production in Europe: National Centre for Nuclear Research Radioisotope Centre Polatom, Poland; Curium Radiopharmaceutical Production, Paris, France, and ITG Radiopharmaceutical production, Garching, Germany. Curium is the world leader in radiopharmaceutical's production formed by the union of the two main radiopharmaceutical companies from USA and Europe: Mallincrodt Nuclear Medicine and IBA Molecular. Meeting was organized with the aim to discuss possibilities of a steady supply of quality diagnostic and therapy solutions especially for inoperable tumors with the highest standards of quality and safety assured at every stage. The conclusion was that radiolabelled magnetic nanoparticles had great potential for neuroblastoma and other less radio-sensitive tumour due to synergistic effect of both hyperthermia and radionuclide therapy.

Project Webpage and dissemination to broader public - Setting-up and permanent updating of the MagBioVin webpage ensures the access to all relevant information on the ongoing activities and achieved results to specialists in the field, as well as to a broader range of potential stakeholders. A direct and visible link to the MagBioVin webpage is posted on the webpage of the VINCENT Center of Excellence, which is a constitutive unit of the “Vinca” Institute. Besides information on the ongoing project activities this webpage will be enriched with the following contents: search engine, links to major relevant scientific and industrial institutions, and electronic newsletter. A special section of the MagBioVin webpage is devoted to a broader public where information is provided in non-scientific language. In addition, a brief printed brochure has been prepared in a similar fashion to provide dissemination among broader public. In addition to internet presentation, fruitful contact with the local media has been achieved so that main project ideas and its achievements appeared in one newspaper article, two TV shows, and a short movie about “Vinca” Institute prepared for its 70 years anniversary celebration in 2018.

B.2. Exploitation of results
Main scientific goal of the MagBioVin project is standardization of methodology for production of activated and radiolabeled MNPs for their use in diagnostic and therapeutic medical protocols. This comprehends several stages from the controlled synthesis and characterization ( physicochemical, biocompatibility and biodistribution, toxicity) to efficiency in diagnostic and therapeutic applications. Such a wide palette of properties enables a multiple fields for exploitation of achieved results.

One of the MagBioVin outcomes will be the ability of the project research team to produce tailor-made biocompatible MNPs. This will have a significant impact on research teams from other R&D institutions in the country working in the area of biomedical applications. This comprehends biochemical and medical institutions working on cell separation, magnetic resonance imaging, cancer diagnostic and therapy, etc. Instead of relying on the commercially available products imported from the abroad, they will have the possibility to express their specific needs and to participate in tailoring of the MNPs–based biocompatible material suitable for targeted application. In addition, this will cultivate a synergy of basic and applied science disciplines, and raise the awareness of potential stakeholders on the increased research abilities of national R&D centres.

Radiopharmaceuticals developed and produced in the “Vinca” Institute (LRVIN Laboratory) are already in regular use in major medical centres in Serbia (Nuclear Medicine Institute of the Serbian Clinical Centre in Belgrade; Nuclear Medicine Institute of the Military-medicine Academy in Belgrade; Oncology and Radiology Institute of Serbia in Belgrade; Oncology Institute of Vojvodina in Sremska Kamenica), as well as in other countries from the region (Bosnia and Herzegovina – Clinical Center Banja Luka, and FYRO Macedonia). Present level of production and quality control are already of high standards, however future training in EU centres dealing with radiopharmaceutical production (CERN, Switzerland; Demokritos, Greece) will bring further improvements by implementation of good manufacturing practice (GMP) in accordance with high EU standards.

The long-term collaboration of MagBioVin team with medical institutions will be of substantial importance in exploitation of result and transferring of knowledge from the MagBioVin project to end-users. In addition, the existing insight into the research potentials of medical institutes in the country/region will help in identifying the most promising centres for future collaborative work on the specific medical applications of MNP. In this way the longer-term impact of the MagBioVin project are expected to reach the clinical practice.

Among important project target group for exploitation of results comprehends pharmaceutical companies from Serbia. After the privatization process that took place in Serbia during the last decade majority of these companies are now parts of international corporations: for instance “Zdravlje” (Leskovac, Serbia) is a member of the Actavis International Group (www.actavis.com) while “Hemofarm” (Vrsac, Serbia) is a part of the Hemopharm Group (www.hemopharm.com). The expected outcome of the MagBioVin project is the know-how for highly specialized manufacturing of pharmaceuticals based on MNPs and radiolabel-MNPs. Transfer of this knowledge to pharmaceutical companies in the country will raise their competitiveness not only in the region but also in the EU market being part of large scale international corporations.

One of the MagBioVin stakeholders is the SME "nB Nanoscale Biomagnetics" from Spain. This company is dealing with the design, development and construction of biomedical instrumentation. It’s DM Series of field applicators and magnetic hyperthermia accessories are the first technological solution in magnetic hyperthermia available in the market. This company was the vendor of the hyperthermia device for the MagBioVin project. They expressed strong interest into orientation of the MagBioVin team towards application of MNPs in magnetic hyperthermia since this is one of the most important area covered by their production and development program. They agreed to provide expert consultancy during MagBioVin training activities concerning magnetic hyperthermia research. In return, the usage of their device in the research activities on magnetic hyperthermia will provide useful feedback for their R&D team and thus help in the future development of similar devices. In addition, DM2 will be the first device of this type in the WB region so it will represent an important reference for future entering of this company into the WB market.
Thus involvement of SME from EU and national pharmaceutical companies which are part of large international companies as project stakeholder will provide spreading of the MagBioVin impact from WB onto EU scientific and economic region.

The improvement of the management skills in the "Vinca" Institute is an important foreseen outcome of the project. This concerns not only the management of the complex multidisciplinary research teams but also exploitation of end-results regarding transfer of technology to industryand private sector, intellectual property (IP) management, licences, etc. These management skills are underdeveloped not only in the country but also in WB region in general. MagBioVin project contains several measures to overcome this drawback in "Vinca", such as a workshop devoted to IP management, visit of management experts from EU scientific institutions for lectures and discussions, and adoption of know-how in this area during the stays in partner institutions from EU. Project Management Board was also a participant in the initiative on opening of the position in "Vinca" for technology transfer and IP management within the already existing Office for International Collaboration. This will serve as an initial seed for speeding these practice into another institutions of similar type in the country. Moreover, being the largest research institute in WB region, "Vinca" will serve as an example to others in the region how to protect, licence and spin-out the technology, and spread awareness about importance of defining the IP rights distribution at the beginning of a scientific or R&D project. The awareness about IP and innovation potential will be continuously increased in WB region through workshops and meetings with representatives of research institutions, policy makers and stakeholders.

List of Websites:
Public Website of the project: www.vincent.org.rs
Era Chair holder: Dr Zeljko Prijovic, email: prijovic@vinca.rs
Project Coordinator: Dr Bratislav Antic, bantic@vinca.rs