Project title: "Interdisciplinary research on multifunctional hybrid particles for biorequirements” acronim: INTERBIORES Contract no. 211/2012 Project.

Project title:"Interdisciplinary research on multifunctional

hybrid particles for biorequirements”acronim: INTERBIORES

Contract no. 211/2012

Project Cod: PN-II-PT-PCCA-

2011-3.2-0428 – Programme PN II –

Partnerships Priority Areas -

Applied Research Projects –

Type 2

Contracting Authority: Executive Unit for Financing HigherEducation, Research Development and Innovation (UEFISCDI)Contractor: "Petru Poni" Institute of Macromolecular Chemistry,Iasi

Duration of project: 02.07.2012 – 02.07.2015 The total value of the contract 3.323.334 lei From the source of funding:

Source 1 - The state budget: 2.950.000 lei

Source 2 - from other sources

(Net financing) 373.334 lei Project Director: CS I Dr. Aurica P. Chiriac

Members:

“Petru Poni” Institute for Macromolecular Chemistry – Iasi

- Project Coordinator

Project Director: Dr. Chiriac P. Aurica “Gr. T. Popa” Medicine and Pharmacy University Iasi –

UMF - Partner 1

Team leader: Dr. Verestiuc Liliana Research and Development National Institute of Technical

Physics Iasi Iaşi - Partner 2

Team leader: Dr. Chiriac Horia SC REZISTOTERM SRL Partner 3

Team leader: Dr. Chiriac Mihai

Abstract:

The project with multi-disciplinary character is focused on analysis and solving from the physical, chemical and biochemical viewpoint the design and technology of novel systems, based on antioxidants-loaded core-shell magnetic nanocomposites deposited onto the stent surface. Novel formulation methods based on the use of biocompatible polymers will be developed and applied to create a family of magnetic nanoparticles (MNPs) further characterized as a platform for magnetically guided delivery of therapeutics. The selected polymers will undergo physical (forming of interpolymer complexes by physical interactions) and chemical (functionalization, derivatization, crosslinking, reactive mixing) modifications in order to obtain stable multifunctional nanosystems. The key features of the new target delivery systems will be investigated, including in vitro bioactive compounds activity, capacity to protect the antioxidants from proteolysis, as well as the capacity of the magnetic guidance and retrieval. The project includes aspects like toxicology, biocompatibility of the nanodevices, and also efficacy and biodistribution of the system. The studies are doing to the facts that a major problem associated with target delivery is the inability to deliver pharmaceuticals to a specific site of the body without causing nonspecific toxicity. The bioproducts loaded magnetic nanoparticles have several advantages such as: small particle size, large surface area, magnetic response, biocompatibility and non-toxicity and are directed with external magnets to the right site, and requires smaller dosage because of targeting, with no side effects.

One of the most innovative aspects of this proposal is the use of functionalized magnetic nanoparticles with antioxidative biomolecules deposited onto the stent surface to realize a drug-eluting-stent type for bio-requirements. The new stent device will functioning as a delivery platform. At the same time, the prepared MNPs will represent a particularly appropriate tool based on their ability to be simultaneously functionalized and guided by an external magnetic field, the presence of the antioxidative biomolecules would be an additional benefit.

To conclude, the purpose of the multi-disciplinary character project is to realize target delivery systems based on hybrid bio-nano-composites with improved magnetic performance of the nanoparticles and maximized therapeutic potential of the drug eluting/retrieval stents by the loaded antioxidative biomolecules layered on the stent surface. In addition, the design of the nanoparticles will include the improvement of the monodispersity, colloidal stability and functionality. Also, further engineering of these nanoparticles and of their formulation as hybrid systems for target delivery will allow improving their bioselectivity and bioefficiency.

Concept and objectives:

The project with multi-disciplinary character is focused on analysis and solving from the physical, chemical and biochemical viewpoint the design and technology for the stents achievement with drug delivery possibilities, based on antioxidants-loaded core-shell magnetic nanocomposites deposited onto the stent surface.

The market for stents is, in many ways, still emerging. While coronary stents have been on the commercial market in one form or another for several years, the technologies and materials used to create the devices are improving every day. In addition, innovative technology is bringing new classes of devices (e.g., fully degradable stents) to market, technologies that grow the market and even expand means of diagnosis and therapy stent to new patient populations in some cases.

One of the most innovative aspects of this proposal is the use of functionalized magnetic nanoparticles with antioxidative biomolecules deposited onto the stent surface to realize stent devices type for bio-requirements. Thus the new stent device will function as a delivery platform. At the same time, the prepared MNPs will represent a particularly appropriate tool based on their ability to be simultaneously functionalized and guided and/or removed by an external magnetic field owing to the magnetic NPs inclusion, meanwhile the presence of the antioxidative biomolecules would be the additional benefit. As it is well known stents are scaffoldings, usually cylindrical or tubular in shape, which function to physically hold open and, if desired, to expand the wall of the vessel. Typically stents are capable of being compressed, so that they may be inserted through small cavities via catheters, and then expanded to a larger diameter once they are at the desired location. Although stents are significant innovations in the treatment of occluded vessels, there remains a need for administering therapeutic substances to the treatment site. Systemic administration of the therapeutic substance often produces adverse or toxic side effects for the patient. Local delivery of therapeutic substances, by contrast, provides a smaller overall dosage that is concentrated at a specific treatment site. Local delivery can produce fewer side effects and achieve more effective results. In this context novel formulation based on biocompatible polymers will be developed and applied to create a family of antioxidant magnetic nanoparticles (MNPs) for covering the stent surfaces further characterized as a platform for magnetically guided and delivery of therapeutics. The selected polymers will undergo physical (forming of interpolymer complexes by physical interactions) and chemical (functionalization, derivatization, crosslinking, reactive mixing) modifications in order to obtain stable multifunctional nanosystems. The key features of the new target delivery systems will be investigated, including in vitro bioactive compounds activity, capacity to protect the antioxidants from proteolysis, as well as the capacity of the magnetic guidance, retrieval and remove. The project includes aspects like toxicology, biocompatibility of the nanodevices, and also efficacy and biodistribution of the system. In addition, the design of the nanoparticles will include the improvement of the monodispersity, colloidal stability and functionality. Also, further engineering of these nanoparticles and of their formulation as hybrid systems for target delivery will allow improving their bioselectivity and bioefficiency.

Stage I/2012 – the obtained results:

In accordance with the plan of the project "Interdisciplinary research on multi-functional hybrid particles for bio-requirements", the stage I/2012 has the objectives:

(I) magnetic composites preparation and (II) their physical – chemical evaluation, which were fulfilled by specific activities : - the obtaining of magnetic nanoparticles (NPs) with linking capacity and antioxidant enzymes transport; - the surface modification of magnetic NPs with polymeric structures, as basis for antioxidant hybrid materials; - the characterization of the magnetic composites with the aim of association with antioxidant enzymes and the estimation of the physical-chemical theoretical and experimental conditions necessary for their realization, and the dissemination of the results by national and international symposia communications, publications in the ISI quoted journals or indexed in international databases.

The conducted studies have resulted in: (I) Synthesis and characterization of polymeric matrices, respectively: (1) poly(2-hidroxyethyl methacrilate-co-3,9-divinyl-2,4,8,10-

tetraoxaspiro[5.5]undecan-co-glicidil methacrylate) ternar copolymer (synthesized by radical copolymerization in aqueous dispersion); 2) poly(dimethyl acrylamide-co-3,9-divinyl-2,4,8,10-tetraoxaspiro [5.5]undecan-co-itaconic acid) ternar copolymer (synthesized by

radical copolymerization in dimethyl acetamide solution); (3) poly(2-hydroxyethyl methacylate-co-3,9-divinyl-2,4,8,10-tetraoxaspiro [5.5]undecan –coacrylamide)(synthesized by radical

copolymerization in dimethyl acetamide solution); (4) 6 variants of bloc copolymers based on poly(succinimide)-b-poly(ethylene glycol)(PEG) (synthesized by copolycondensation in

dimethyl formamide in the presence of Mn(CH3COO)2.2H2O as catalyst ), PEG having different molecular weights, such as : 2000, 3000, 4000, 10000, 20000 and 35000;

(5) one grafted polymeric structure based on carboxymethyl starch-g-poly(lactic acid); (II) The preparation of hybrid structures with magnetic characteristics by using the realized polymeric matrices; (III) Testing activities of the polymeric matrices and hybrid structures for coupling of the antioxidant enzymes, in course.

In the context of the dissemination activity of the results by communications at national and international symposia, by publications in the ISI quoted journals or indexed in international databases, there were presented:

(a) Three communications at 5th International Conference “Biomaterials. Tissue Engineering and Medical Devices” BiomMedD’2012, Constanta, 29 August – 1 September 2012, respectively:

Streptavidin-Biofunctionalized Magnetic Particles for Blood Contacting Applications; V. Bălan, M.I. Popa, A.P. Chiriac, I. Neamtu, L.E. Nita, M.T. Nistor, M. Butnaru, L. Verestiuc

Bioactive Hybrid Scaffolds in Regenerative Medicine and Tissue Engineering; M.T. Nistor, C. Zgardan, C. Vasile, L.E. Nita, A. Chiriac

Assembly Design and Characterization of an Innovative Modulated Drug Delivery System; L. Nita, M. Nistor, N. Tudorachi, I. Neamtu, A. Chiriac

(b) One communication „Determination of the kinetic parameters and analysis of gases released by thermal decomposition of CMS-g-PLA copolymer” at XXXII National Chemical Conference Rm Valcea, October 2012, and

( c ) One paper accepted for publication in Industrial & Engineering Chemistry Research (IF = 2.237): “Thermal degradation of carboxymethyl starch-g-poly(lactic acid) copolymer by TG-FTIR-MS analysis”, authors Nita Tudorachi, Rodica Lipsa, Fanica Mustata.

Stage II/2013:

In accordance with the plan of the project „Interdisciplinary research on multi-functional hybrid particles for bio-requirements”, the phase II/2013 had as objectives (I) Magnetic composites synthesis and (II) Physical-chemical evaluation (phase I/2012 continuation). The objectives were carried out through and finalized by specific activities, such as:

- the obtaining of magnetic nanoparticles (NPs) with linking capacity and antioxidant enzymes transport;- experimental development for magnetic NPs surface modification with the aim of obtaining molecular adapted bio-structures, as

basis for new hybrid materials;- the evaluation of antioxidant enzymes, significant for biological structures;- magnetic NPs bio-functionalization with biological structures for the nanometric level control of the biological and biochemical

processes;- experimental development for the obtaining of bio-functionalized magnetic NPs;- magnetic composites characterization in association with antioxidant enzymes for the estimation of the experimental and theoretical

physical-chemical conditions necessary for their preparation;- elucidation and characterization of the surface properties of the particles, associated with antioxidant enzymes coupling reactions

and results interpretation for subsequent studies of targeted delivery and controlled release;- dissemination of the results by communications at national and international symposia, by publications in the ISI quoted journals or

indexed in international databases.- The dissemination of the results by communications at national and international symposia, by publications in the ISI quoted

journals or indexed in international databases, materialized as it follows:

The obtained results:

Published papers:

- Characterization of the semi-interpenetrated network based on collagen and poly(N-isopropyl acrylamide-co-diethylene glycoldiacrylate). Authors: MT Nistor, A.P. Chiriac, LE Nita, C Vasile. Int J Pharmaceutics 452 (2013) 92– 101; IF=3.458.

- Multilayered structure based on poly(N,N-dimethyl-acrylamide-co-3,9-divinyl-2,4,8,10-tetraoxaspiro (5.5) undecane) prepared in a multiphase gelation process. Authors: AP Chiriac, LE Nita, MT Nistor, L Tartau. Int J Pharmaceutics 456 (2013) 21– 30; IF=3.458.

- Obtaining of new magnetic nanocomposites based on modified polysaccharide. Authors: NTudorachi, AP Chiriac, Carbohydrate Polymers 98 (2013) 451– 459; IF=3.479.

- Upon the Delivery Properties of a Polymeric System Based on Poly(2-Hydroxyethyl Methacrylate) Prepared with Protective Colloids. Authors: LE Nita, AP Chiriac, M Nistor, TBudtova. J Biom Nanobiotechnol 4(2013), 357-364; ISSN Online: 2158-7043.

- Upon the Developments of Drug-Eluting Stents in the Treatment of Coronary Lesions. Authors: A.Diaconu, V Balan, AP Chiriac. Recent Patents on Materials Science 6(2013) 229-237; ISSN:1874-4648.

- Semi-interpenetrated Network with Improved Sensitivity Based on Poly(N-Isopropylacrylamide) and Poly(aspartic acid). Authors: MT Nistor, AP Chiriac, LE Nita, INeamtu, CVasile. Polym Eng Sci 53(2013) 2345-2352; IF=1.243.

- Poly(N,N-dimethylacrylamide-co-3,9-divinyl-2,4,8,10-tetraoxaspiro(5.5)undecane) synthesis as matrix ensuring intramolecular strategies for further coupling applications. Authors: AP Chiriac, MT Nistor, LE Nita, I Neamtu. Rev. Roum. Chim. 58(2013)(2-3), 129-136; IF-0.331.

The obtained results:

- Hydrogel based on poly(N,N-dimethylacrylamide-co-3,9-divinyl-2,4,8,10-tetraoxaspiro(5.5)undecane) with dual sensitive behavior. Synthesis and characterization. Authors: LE Nita, AP Chiriac, MT Nistor, I Neamtu. Rev. Roum. Chim., 2013, 58(2-3), 137-143; IF-0.331.

- Poly(acrylic acid)/ poly(ethylene glycol) nanoparticles designed for ophthalmic drug delivery. Authors: A.M. Vasi, M.I.Popa, E.C.Tanase, M. Butnaru, L. Verestiuc, agreed to be published in J. Pharm. Sci.; IF=3.13.

Book Chapters :- Chapter 3. Hybrid Sensitive Hydrogels for Medical Applications. MT Nistor, C Vasile, AP Chiriac, A Rusu, C

Zgardan, LE Nita, I Neamtu. In: Polymer materials with smart properties. Ed. M Bercea. Nova Science Publ. New York 2013, 67 – 89.

- Chapter 8. Sol-Gel Technique Implemented for Biomedical Applications. LENita, AP Chiriac, I Neamtu. 189-204. In: Polymer materials with smart properties. Ed. M Bercea. Nova Science Publ. New York 2013, 189 – 204.

Published papers in Proceedings:- Functionalized superparamagnetic nanoparticles as versatile carriers for targeted antioxidant enzyme therapy .

Authors: V.Balan, M. Butnaru, O. Bredetean, L. Profire, G. Lupascu, A.P.Chiriac, L.E. Nita¸ I. Neamtu, L.Verestiuc Proceedings of the 4th ed. of E-Health and Bioengineering Conference-EHB 2013, Iasi, Romania.

- Biomimetic composites based on calcium phosphates and chitosan - hyaluronic acid with potential application in bone tissue engineering. Authors: F.D.Ivan, A.Marian, C. E. Tanase, M. Butnaru, L. Vereştiuc, Bioceramics 25, Key Engineering Materials, 587, 191-196.

The obtained results:

Communications at scientific meetings:- Functionalized superparamagnetic nanoparticles as versatile carriers for targeted antioxidant enzyme therapy.

V. Balan, M. Butnaru, O. Bredetean, L. Profire, G. Lupascu, A.P.Chiriac, L.E. Nita¸ I. Neamtu, L.Verestiuc, 4th IEEE International Conference on E-Health And Bioengineering - EHB 2013, Iaşi, Romania, November 21st-23rd, 2013.

- Biomimetic composites based on calcium phosphates and chitosan - hyaluronic acid with potential application in bone tissue engineering. 25th Symposium and Annual Meeting of the International Society for Ceramics in Medicine; Bucharest, Romania November 07-10th 2013; Autori: F.D.Ivan, A.Marian, C. E. Tanase, M. Butnaru, L. Vereştiuc, Bioceramics 25, Key Engineering Materials, 587, 191-196.

  Poster at scientific meetings:- Process for magnetic composites synthesis. Authors: AP Chiriac, LE Nita, INeamtu, MT Nistor. The 17th

International Salon of Research, Innovation and Technological Transfer “Inventica 2013” 19 – 21 st June 2013 Iasi – Romania.

- Hybrid Structures for Bioapplications. Authors: AP Chiriac, LE Nita, INeamtu, MT Nistor. National Innovation Salon CHIM-INVENT, 3 – 5 July 2013, Iasi Romania.

Registered patent application: - Magnetic composite synthesis process. Authors : A.P. Chiriac, L.E. Nita, I. Neamtu, N.Tudorachi, A. Diaconu,

V. Balan, C. Munteanu. Patent application No. A 00833/13.11.2013.

Stage III/2014: Plan and activities

Physico-chemical evaluation (continuation from 2013) Bio-chemical evaluation; Methods for toxicological evaluation of NPs for providing fundamental knowledge on their

design and testing as targeted delivery systems. Integration of bio-magnetic systems

- Activity 2.7

Characterization of magnetic composites in combination with antioxidant enzymes to determine the

experimental conditions to achieve them - continuing in 2012 and 2013

- Activity 2.8

The elucidation and characterization of surface properties of the particles associated with the coupling reaction

of the antioxidant enzyme for further studies of release (activity continuation from 2012 and 2013)

- Activity 2.9

Determination of the initiation reactions of self-assembling mechanisms for hybrid materials preparation - Part

- Activity 2.10

Conceptual design of coupling system between the magnetic composite and the antioxidant enzyme -Part I

- Activity 2.11

Dissemination of results through communications at national and international symposia, publications in

journals

Stage III/2014: Plan and activities

- Activity 3.1

Establish procedures for the preparation and characterization of new materials

- Activity 3.2

Dissemination of results through communications at national and international symposia, publications in

journals ISI or indexed in international databases

- Activity 3.3

Testing biomagnetic self-assembled hybrid materials with antioxidant characteristics - Part I

- Activity 3.4

Testing and evaluation of hybrid systems - Part I

The obtained results:

Published papers:1. Current concepts on cardiovascular stent devices; I. Neamtu, A. P. Chiriac, A. Diaconu, L.

E. Nita, V. Balan, M. T. Nistor; Mini-Reviews in Medicinal Chemistry, 14, 505-536 (2014). If=3.186

2. Semi-imprinting quercetin into poly[N,N-dimethylacrylamide-co-3,9-divinyl-2,4,8,10-tetraoxaspiro (5,5) undecane] network: Evaluation of the antioxidant character; A. P. Chiriac, L. E. Nita, L. Tartau, I. Neamtu, M. T. Nistor; Journal of Pharmaceutical Sciences, 103, 2338-2346 (2014). IF=3.007

3. Upon some multi-membrane hydrogels based on poly (N,N-dimethyl-acrylamide-co-3,9-divinyl-2,4,8,10-tetraoxaspiro (5.5) Undecane): preparation, characterization and in vivo tests, L. E. Nita, A. P. Chiriac, M. T. Nistor, L. Tartau; J. Mat. Sci.: Mat. in Med., 25 (7), (2014) 1757-1768, IF=2.379.

4. Upon Synthesis of Poly(N-isopropylacrylamide-co-2-dimethyl-aminoethyl methacrylate-co-itaconic acid) Copolymers as Matrix Ensuring Intramolecular Strategies for Further Coupling Applications; A. P. Chiriac, L. E. Nita, I. Neamtu, V. Balan, A. Diaconu; Journal of Research Updates in Polymer Science, 3 (1), 48-56 (2014). IF-0.0

The obtained results:

Communications at scientific meetings:1. A. Diaconu, A. P. Chiriac, L. E. Nita, N. Tudorachi, I. Neamtu, V. Balan; Upon synthesis of poly(maleic

anhydride-co-3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane) copolymer with antioxidant character and possibilities for bioconjugation; communicated at 2nd International Conference on Chemical Engineering, Iasi , November 5-8 2014.

2. A. Diaconu, I. Neamtu, L. E. Nita, A. P. Chiriac, V. Balan, Poly(maleic anhydride-co-3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane) copolymer-based magnetic composites: Potential vectors for remote-controlled bioactive substance release, 2nd International Conference on Chemical Engineering, Iasi , November 5-8 2014.

3. Tudorachi, A. P. Chiriac, G. Lisa, V. Balan, L. Nita, Magnetic nanocomposites PLGA/magnetite, “Alexandru Ioan Cuza” University Days, Faculty of Chemistry Conference, Iasi, 31 octombrie – 01 noiembrie 2014,

4. R. Lipsa, N. Tudorachi, A. Grigoras, C. Vasile, P. Gradinariu, A. P. Chiriac, F. Mustata, Poly(vinyl alcohol) copolymers biodegradation with Trichotecium roseum fungi “Alexandru Ioan Cuza” University Days, Faculty of Chemistry Conference, Iasi, 31 octombrie – 01 noiembrie 2014.

Registered patent application: Process for the synthesis of a magnetic composite ; A.P. Chiriac, L.E. Niţă, I. Neamtu, N. Tudorachi, A. Diaconu, V. Balan, A00808 from 29.10.2014.

Stage IV/2015 – Project objectives

Physical – Chemical evaluation – continued 2014 Stage Biochemical evaluation; Methods for toxicological evaluation of new NPs to provide fundamental

knowledge on design and testing as targeted delivery systems Integration of bio-magnetic system – continued 2014 Stage Activity 2.9 . Determination of the initiation mechanism of the self-assembling reaction of hybrid materials –

Part III Activity 2.10. Conceptual approach and the design of the coupling system between antioxidant magnetic

composite and stent – Part IIIActivity 3.3. Testing of self-assembled biomagnetic hybrid materials with antioxidant characteristics. Part II.

Activity 3.4. Hybrid systems testing and evaluation – Part III Activity 4.1. Working methods and protocols for new therapeutic strategies, applications and techniques for

testing of the obtained hybrid systems. Toxicological evaluation methods of the new synthesized nanoparticles for testing them as targeted delivery systems.

Activity 4.2. Developing the capacity of knowledge of particle / stent systems and transport through different specific environments

Activity 4.3. Optimization and synthesis studies. Activity 4.5.1. Dissemination of the results through publications in ISI journals or indexed in international

databases. Activity 4.6. Correlation and optimization of the interdependent functions for optimal conditions of hybrid

magnetic antioxidant structures / stent realization. Developing of the laboratory technology for the integration of new hybrid materials as biomagnetic composites with antioxidant characteristics. Part I

Activity 2.9. Determination of the initiation mechanism of the self-assembling reaction of hybrid materials

Illustrating the initiation of the reaction mechanism of self-assembling hybrid

materials

Making of the polymer coating on the surface of magnetite is based on several physical interactions that lead to a process of assemblying between the two components and creating of a stable complex hybrid system with magnetic characteristics.

The formation of the hybrid type "core (magnetite) - shell (synthetic polymer)" is based on the occurrence of dipole electrostatic interactions, hydrogen bonding, hydrophobic interactions and van der Waals forces that determine the self-assembling process and the system stability.

Meanwhile, the hybrid system has free functional groups for subsequent attachment of new active structures, for example enzymes, which is the generic objective of the project.

For the antioxidant magnetic composite realization by coupling antioxidant enzyme – catalase – with the magnetic composite, it is proceeded to two coupling methods, as shown:

by physical links (reversible)

and by chemical links (irreversible) via

water-soluble carbodiimides:1-ethyl -3-(3-dimethylaminopropyl) carbo-diimide (EDAC)

Illustration of the coupling procedure of antioxidant enzyme

(catalase) with the magnetic composite

Evaluation of the magnetic composite functionalization with antioxidant enzyme

Dimensional distribution (a) and zeta potential distribution (b) of the magnetic composites before and after the functionalization

Dimensional distribution (a) and zeta potential evaluation (b) of the magnetic composite before and after the functionalization with catalase confirm the immobilization of the enzyme on the surface of the magnetic composite.

Activity 2.10. Conceptual approach and the design of the coupling system between antioxidant magnetic composite and stent

The coating procedure of MC nanoparticles to the stent surface is done in an alternating magnetic field (AMF). In designing the system to achieve the conceptual approach of the new process is used the fact that magnetic hybrid nanocomposites as stable dispersed colloidal suspensions in a suitable solvent, can generate heat in AMF and also they show affinity for metal stent. Thus, there are created the right conditions for a uniform deposit on the surface of the stent. It is used an AMF with intensity H = 200 Oe, obtained from a solenoid with the following characteristics: L = 700 μH, V = 125 kHz, I = 2.5 A, U = 1.3 Kv, P = 3 Kvar.

After 10 minutes of maintaining the stent and the magnetic nanoparticles dispersion in AMF, the ambient temperature does not exceed 52 C, that certifies a relatively low Curie temperature for magnetic nanoparticles. It can be concluded that nanoparticles avoid the overheating of the tissue, when they are excited by AMF. Thus, these magnetic nanoparticles can be used for treatments including hyperthermia (when the AMF action is reduced to 2-3 min).

Magnetic susceptibility evaluation and the film morphology deposited on metallic stent device

Optical microscopy imaging of the magnetic composite coated stentMagnetic susceptibility values confirm the presence of magnetic composite nanopar-ticles on the stent surface.

Deposition cycle

Stent weight, g Stent magnetic susceptibility, e4 V

Before deposition

After deposition

Before deposition

After deposition

- 0.0120 - 0.023 -

I - 0.0140 - 0.052

II - 0.0142 - 0.054

III - 0.0146 - 0.058

IV - 0.0150 - 0.069

- After 5 repeated washings

- - 0.0130 - 0.036

Activity 3.3. Testing of self-assembled biomagnetic

hybrid materials with antioxidant characteristics

20 40 60 80 100 1200,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

Witness PMAU PMAU_E 1/1 PMAU_E 1/3 PMAU_E 1/4 PMAU_E 1/5

Ab

sorb

an

ce, 5

17

nm

Time, min

1000 1500 2000 2500 3000 3500 4000 4500

0,4

0,5

0,6

0,7

0,8

0,9

1,0

Ab

sorb

an

ce, 5

17

nm

Time, min

Witness PMAU PMAU_E 1/1 PMAU_E 1/3 PMAU_E 1/4 PMAU_E 1/5

The new polymer synthesized structures are evaluated in terms of antioxidant character by radical reactions in the presence of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical.

28% inhibition of DPPH radicals in case of functionalized PMAU copolymer with meso-erythritol PMAU_E (1/5 ratio PMAU / erythritol) is recorded, meanwhile erythritol witness exhibited 29 % inhibition and the PMAU copolymer just 14.75% reduction. It can be concluded that the antioxidant ability of the functionalized copolymer is conferred by erythritol, but at the same time the PMAU copolymer has antioxidant character. It is noticeable that the antioxidant character is manifested by the system PMAU_E 1/5 even after 72 hours testing period.

Activity 4.1. Working methods and protocols for new therapeutic strategies, applications and techniques of testing for the obtained hybrid systems

The results of investigation of acute toxicity and biocompatibility of the magnetic composites

According to Hodge & Sterner toxicity scale is observed that the value LD50 is in the range of 500 - 5000 mg / kbw, allowing the placement of MC in the group of low toxic substances.

There are no significant variations between the values of the elements of blood leukocyte formula for mice in the treated group with MC compared to the control group treated with distilled water, for 24 hours and 14 days. The laboratory analysis present no significant differences for glutamic pyruvic transaminase (GPT), glutamic oxalacetic transaminase (GOT) and lactic dehydrogenase (LDH) activities between the control and the treated MC groups, after 24 hours and after 14 days from the i.p. administration. In terms of serum opsonic capacity (OC), the phagocytic capacity (FC) and bactericidal capacity (BC) of peritoneal macrophages for the control group treated with distilled water and the treated group with MC, there are no major variations in the results.

Toxicological evaluation methods of the new synthesized nanoparticles for testing them as targeted delivery systems

Microscopic image of liver structure: a - normal control group; b - with diffuse liver stasis and liver regeneration in the group treated with MC (Van Gieson staining, x 40)

Acute toxicity investigation shows that MC particles are relatively harmless from the toxicological point of view when i.p. administered to mice. They exert similar changes on leukocyte formula items and in enzymatic activity like the distilled water administered by i.p. injection in control group. MC administration does not alter the animal's immune defense capability compared with the control group. The major histopathological changes do not occur in the liver structure. The results suggest good biocompatibility by in vivo tests in mice after administration of MC.

Optimization and synthesis studies

O

O

O

O

O

O

O

O

O

O

In the attempts to synthesize a polymer matrix having the best features for obtaining a magnetic composite, but with functional versatility for further coupling of biologically active substances (enzymes), the synthesis, properties and sensitive behavior of a set of random copolymers based on itaconic anhydride (IA) with various molar ratios of 3,9-divinyl-2,4,8,10-tetraoxaspiro [5.5] undecane (U) in the structure, are followed.

Poly(itaconic anhydride-co-3,9-divinil--2,4,8,10-tetraoxaspiro [5,5] undecan)

– schematic structure-

These new polymer structures, because of their ability to form networks, to possess excellent biodegradability and biocompatibility, amphiphilicity, oxidative properties and good thermal stability, they are good film formers, sensitive to acidic pH, but also with binding opportunities required by further investigations. This behavior can be explained by random spiral-type conformation of these copolymers, with folded and packaged macromolecules by physical interactions occurring between chains. The spiroacetal segment with acidic pH sensitivity is capable of interaction from the ether oxygen with other functional groups by coordinative or hydrogen bonding, and thus to induce the dynamic change of macromolecular chains’ stereochemistry through anomerically effect, interfered with temperature increasing in the thermal agitation conditions.

Dissemination of the results

Published papers•Design and synthesis of a new polymer network containing pendant spiroacetal moieties; authors: A. Diaconu, A. P. Chiriac , L. E. Nita, N. Tudorachi, I. Neamtu, C. Vasile, M. Pinteala; Designed Monomers and Polymers, 2015, 18(8), 780-788, IF=2.78•New nanocomposite based on poly(lactic-co-glycolic acid) copolymer and magnetite. Synthesis and characterization; authors: N. Tudorachi, A. P. Chiriac, F. Mustata; Composites Part B-Engineering, 2015, 72, 150-159, IF=2.983.•Synthesis and thermal investigation by TG-FTIR-MS analysis of some functionalized acrylic copolymers and magnetic composites with Fe3O4; authors: N. Tudorachi, I. Bunia; J. Anal. Appl. Pyrolysis, 2015, http://dx.doi.org/doi:10.1016/j.jaap.2015.09.010, IF=3.56.•Degradation of poly(vinyl alcohol)-graft-lactic acid copolymers by Trichotecium roseum fungus; authors: R. Lipsa, N. Tudorachi, V. C. Grigoras, C. Vasile; J. Appl. Polym. Sci. 2015, 132, 14. (DOI: 10.1002/APP), IF=1.77.•Static and dynamic investigations of poly(aspartic acid) and Pluronic F127 complex prepared by self-assembling in aqueous solution; authors: L.E. Nita, A. P. Chiriac, M. Bercea, MT. Nistor; Applied Surface Science, 2015, 359, 486–495, IF=2.711.•Possibilities of quercetin insertion into poly(N, N-dimethylacrylamide-co-3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane) network; authors: A. P. Chiriac, L.E. Nita, I. Neamtu; Materials Science and Engineering C, 2015,47, 17–25, IF=3.088.•In situ preparation of a magnetic composite during functionalization of poly[maleic anhydride-co-3,9- divinyl-2,4,8,10-tetraoxaspiro(5.5)undecane] with erythritol; authors: I. Neamtu, A. P. Chiriac, L.E. Nita, N. Tudorachi, A Diaconu; J Nanopart Res, 2015, 17:254, IF=2.184.•An investigation on multi-layered hydrogels based on poly(N, N-dimethylacrylamide – co – 3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane); authors: A.P. Chiriac, M. T. Nistor, L.E. Nita; Rev. Roum. Chim., 2014, 59(11-12), 1059-1068, IF=0.311•Patterning poly(maleic anhydride-1 co-3, 9-divinyl-2, 4, 8, 10- tetraoxaspiro (5.5) undecane) copolymer bioconjugates for controlled release of drugs; authors: L.E. Nita, A.P. Chiriac, L. Mititelu-Tartaua, E. Stoleru, F. Doroftei, A. Diaconu; International Journal of Pharmaceutics, 2015, 493, 328-40, IF=3.65.•Upon synthesis of a polymeric matrix with pH and temperature responsiveness and antioxidant bioactivity based on poly(maleic anhydride-co-3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5] undecane) derivatives; authors: A. P. Chiriac, L. E. Nita, N. Tudorachi, I. Neamtu , V. Balan, L. Tartau; Materials Science and Engineering C 2015, 50, 348–357, IF=3.088.

Dissemination of the results

Published paper in Proceedings: Study on poly(vinyl alcohol) copolymers biodegradation; authors: R. Lipşa, N. Tudorachi, A. Grigoraş, C.

Vasile, P. Grădinariu; Memoirs of the Scientific Sections of the Romanian Academy, Tome XXXVIII, 2015, 1-22.

Communication at scientific meeting: Biocompatible magnetic nanoparticles with antioxidant enzymes for cardiovascular applications; authors:

L. Lungoci, V. Balan, M. Butnaru, C. Dimitriu, L. Verestiuc; 6th International Seminar on Biomaterials & Regenerative Medicine, Oradea, România, 17-19th September 2015.

Registered patent application: Process for synthesis of a copolymer matrix for biomedical applications; authors: AP Chiriac, LE Nita, A

Diaconu, I Neamtu, N Tudorachi, V Balan, Patent Application No. A/00341/15.05.2015

Dissemination of the results

Posters at scientific meetings

•Upon synthesis of a comprehensive copolymer network based on itaconic anhydride and 3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane for bioconjugation; authors: A. Diaconu, L. Niță, A. Chiriac, E. Butnaru, N. Tudorachi, C. Vasile, M. Pinteala; Frontiers in Macromolecular and Supramolecular Science, Simpozionul Internaţional Cristofor I. Simionescu , VII-th Edition. •Smart polymeric systems based on n,n dimethylacrylamide and 3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane, V. Bălan; authors: A. Diaconu, M. Asăndulesa, E. Butnaru, L. E. Niţă, I. Neamţu, N. Tudorachi, A. P. Chiriac. Zilele Academiei iesene, ed. XXV, 24-26 sept. 2015•SOD and catalase immobilization onto superparamagnetic nanoparticles for cardiovascular applications; authors: L. Lungoci, V.Balan, M. Butnaru, O.Bredetean L.Verestiuc; 12th International Conference on Nanosciences & Nanotechnologies (NN15), 7-10 July 2015, Thessaloniki, Greece. •Antioxidant enzymes immobilized on biocompatible SPIONS for cardiovascular applications; authors: L. Verestiuc, L. Lungoci, V. Balan, M. Butnaru, O. Bredetean; 27th European Conference on Biomaterials, 30 august-3 sept. 2015, Kracovia, Poland.•Investigation upon a novel poly(maleic anhydride – co - 3, 9 – divinyl - 2,4,8,10-tetraoxaspiro (5.5) undecane) / magnetite hybrid nanocomposite preparation; authors: I. Neamtu, A. P. Chiriac, L. E. Nita; Fourth International Conference on Multifunctional, Hybrid and Nanomaterials 9-13 March 2015, Sitges, Spain.•Possibilities for Quercetin insertion into poly(N,N-dimethylacrylamide-co-3,9- divinyl - 2,4,8,10-tetraoxaspiro (5.5) undecane) network; authors: A. P. Chiriac, L. E. Nita, I. Neamtu; Fourth International Conference on Multifunctional, Hybrid and Nanomaterials 9-13 March 2015, Sitges, Spain.