”Petru Poni” Institute of Macromolecular Chemistry Grigore Ghica Voda Alley No 41 A, Iasi, Romania Project No 254/1.10.2015   Code Project: PNII-RU-TE-2014-4-0294 SYNTHESIS OF NOVEL HYDROGELS WITH DEFINED 3D CHARACTERISTICS AND BIODEGRADABILITY PRESENTATION FOR BIOAPPLICATIONS Project leader: LOREDANA ELENA NIŢĂ e-mail: lnazarie@yahoo.co.uk

ABSTRACT The proposed research is a technological approach on obtaining new hydrogels from functional polymeric materials and their use in nanoscale devices. Spontaneous assembly of macromolecular compounds in aqueous medium, extensive exemplified in nature by amphiphilic compounds, is the most promising technological bottom-up approach for obtaining functional materials to be used for encapsulation and controlled release of biologically active molecules in medicine, food and agro-chemistry industry, for adsorption and pre-concentration of toxic organic compounds and heavy metal ions in wastewater treatment and environmental monitoring, or as templates for nano-electronic devices. In this context, we intend to create selective surfaces and interfaces presented by a number of polymers and gels respectively, which will have new features and capabilities.

PURPOSE The project aims to develop new strategies for self assembled complex preparation based on polymers of natural and synthetic origin. The challenge is to have polymer matrix with intrinsic capability for gel formation, stimuli sensitive response, and functionality for coupling specific compounds. The new prepared and investigated systems will be constituted from supramolecular hydrogels formed as result of self-assembly process between a 3D chemically crosslinked macromolecular network structure and another polymeric compound which brings to system supplementary functional groups to improve the coupling capacity, and specific properties as for example biodegradability in specific environment.

Main Objective The main goal of the research project is to prepare novel hydrogels with defined 3D characteristics and biodegradability presentation for bioapplications. This activity will be carried out both from a fundamental as well applied standpoint, using a multidisciplinary approach.

Scientific and Technological Objectives The proposed scientific and technological objectives of this project follow a bottom-up approach involving: 3D polymeric matrix preparation, the fundamental understanding of the supramolecular self-assembly between 3D matrix and a biocompatible/biodegradable polymer, their characterisation in terms of structure vs function using common and advanced techniques, forming derivatives of self assembled structures with specific compounds (bioactive compounds for example) followed by their implementation as hybrid nanostructured materials, as well as their development towards specific applications. At the same time, during the project development will be established (a) the mechanisms for inducing self-assembling at the nanoscale at surfaces and interfaces to give new systems; (b) it will be created macroscale multifunctional interfaces with precise chemical properties, and (c) it will be developed the chemical and physical control for the responsive devices.

Estimative results Design vectors for 3D systems preparation; Preparation of 3D polymeric systems responsible to external stimuli and ability for gelation; Gels preparation on the basis of 3D polymeric systems; Design vectors for self-assembling (SA) systems; Gels preparation on the basis of interpenetrated polymer structures; Characterization of the new polymeric compounds to sustain the design and synthesis of the innovative SA supramolecular systems; Design vectors for hybrid systems preparation; Preparation of hybrid complexes to be used for biomedical applications; Characterization and testing of the prepared hybrid systems.

Phase 1/2015 Synthesis of novel multifunctional macromolecular systems. Preparation of a novel 3D polymeric structure - part 1

Phase 1/2015 Results: (1) Design vectors for 3D system preparation; (2) Preparation of 3D polymeric system responsible to external stimuli and ability for gelation – preliminary phase.

Phase 1/2015 Dissemination: L. E. Nita, A. P. Chiriac, E. Stoleru, A. Diaconu, N. Tudorachi, Tailorable Polyelectrolyte Protein Complex Based on Poly(aspartic acid) and Bovine Serum Albumin, submitted for evaluation

Phase 2/2016 W.P.1. Synthesis of novel multifunctional macromolecular systems. Preparation of a novel 3D polymeric structure - part 2 W.P.2. Creation of new type of self-assembled network structures Preparation of supramolecular systems - part 1

Phase 2/2016 Results: (1) Design vectors for 3D system preparation; (2) Preparation of 3D polymeric system responsible to external stimuli and ability for gelation. (3) Design vectors for SA systems – preliminary phase.

Phase 2/2016 Dissemination Published paper: L. E. Nita, A. P. Chiriac, A. Diaconu, N. Tudorachi,L. Mititelu-Tartau, Multifunctional nanogels with dual temperature and pH responsiveness, International Journal of Pharmaceutics , 2016, 515(1-2):165-175, IF= 3.994. L. E. Nita, A. P. Chiriac, E. Stoleru, A. Diaconu, N. Tudorachi, Tailorable Polyelectrolyte Protein Complex Based on Poly(aspartic acid) and Bovine Serum Albumin, Designed Monomers and Polymers, 2016, 19(7):596-606, IF=1.497. V. Balan, M. Asandulesa, E. Butnaru, A.P.Chiriac, N. Tudorachi, L.E.Nita, I. Neamtu, A. Diaconu, Investigation on the properties of poly (2-hydroxyethylmethacrylate -co- 3,9-divinyl-2,4,8,10-tetraoxaspiro (5.5) undecane) as a functional polymeric system, Rev. Roum. Chim., 2016, 61(8-9), 689-698. IF=0.311.

Phase 2/2016 Dissemination Patent request:   "METHOD OF PREPARATION OF A CROSS-LINKED STRUCTURE WITH BIOMEDICAL APPLICATIONS" - authors: AP Chiriac, LE Nita, A. Diaconu, AG Rusu, I Neamtu, N Tudorachi, Patent request A2016 - 00895/24.11.2016

Phase 2/2016 Dissemination Communication: A.P. Chiriac, L.E. Nita, A. Diaconu, M. Bercea, N. Tudorachi, D. Pamfil, L Mititelu-Tartau, Preparation of a new drug delivery carrier based on hydrogels of hyaluronic acid crosslinked with poly(itaconic anhydride-co-3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane) copolymers, Annual Conference & Expo on Biomaterials―Biomaterials 2016,‖ 14-16 Martie 2016 Londra, UK. A.P. Chiriac, L.E. Nita, N. Tudorachi, L. Mititelu-Tartau, A. Diaconu, M. Bercea,Molecular design of hyaluronic acid gel networks for drug delivery applications,  7th International Conference “Biomaterials, Tissue Engineering & Medical Devices” BIOMMEDD'2016, 15-17 Septembrie 2016 Constanta, Romania. L. E. Nita, A. P. Chiriac, E. Stoleru, A. Diaconu,N. Tudorachi, Tailorability of Poly(Aspartic Acid)/BSA complex by Self-Assembling in Aqueous Solutions, 18th International Conference on Materials Science, Engineering and Manufacturing, ICMSEM 2016, 25-26 Aprilie, Paris, Franta. L. E. Nita, M. Bercea, A. P. Chiriac, I. Neamtu, Viscoelastic Behaviour of Hyaluronic Acid Copolymers, 18th International Conference on Materials Science, Engineering and Manufacturing, ICMSEM 2016, 25-26 Aprilie, Paris, Franta.

Phase 3/2017 . Characterisation of polymers. W.P.2. Creation of new type of self-assembled network structures Preparation of supramolecular systems - part 2. Optimization of SA networks will be carried out by experiments methodology designing. Evaluation of self assembling processes. W.P.3. Characterization and Performance Monitoring Characterisation of polymers. Measuring Cell Viability / Cytotoxicity W.P.4 Inclusion and complexation of specific compounds for the hybrid supramolecular complex preparation Preparation of hybrid supramolecular complexes based on the SA systems and specific compounds Characterisation of hybrid complexes Testing of the hybrid complexes in direct correlation with the nature of the specific compounds .

Phase 3/2017 Results: Design vectors for SA systems. Gels preparation on the basis of the interpenetrated polymer structures. Characterization of the new polymeric compounds to sustain the design and synthesis of the innovative SA supramolecular systems Design vectors for hybrid systems; Preparation of hybrid complexes for application in (a) biomedical applications or for (b) environmental industry; Characterization and testing of the prepared hybrid systems.

Phase 3/2017 Dissemination Published paper: 1. I. Neamtu, A. G. Rusu, A. Diaconu, L. E. Nita, A. P. Chiriac, Basic concepts and recent advances in nanogels as carriers for medical applications, Drug Delivery 24(1) (2017) 539–557, IF=6.4. 2. L.E. Nita, A.P. Chiriac, M. Bercea, M. Asandulesa, B. A. Wolf, Self-assembling of poly(aspartic acid) with bovine serum albumin inaqueous solutions, International Journal of Biological Macromolecules 95 (2017) 412–420, IF=3.671. 3. A.P. Chiriac, L.E. Nita, A. Diaconu, M. Bercea, N. Tudorachi, D. Pamfil, L. Mititelu – Tartau, Hybrid gels by conjugation of hyaluronic acid with poly(itaconic anhydride-co-3,9-divinyl-2,4,8,10 tetraoxaspiro (5.5)undecane) copolymers, International Journal of Biological Macromolecules 98 (2017) 407-418, IF=3.671. 4. A. Diaconu, L.E. Nita, M. Bercea, A.P. Chiriac, A.G. Rusu, D. Rusu, Hyaluronic acid gels with tunable properties by conjugating with a synthetic copolymer, Biochemical Engineering Journal 125 (2017) 135–143, IF=2.892 5. A. G. Rusu, A. Diaconu, N. Tudorachi, M. Asandulesa, L. E. Nita, M. Cristea, I. Neamtu, A. P. Chiriac, Comparative studies regarding the impact of the synthesis possibilities on the physico-chemical properties of poly(n,n-dimethylaminoethyl methacrylate), Rev. Roum. Chim., 62(4-5) (2017) 399-412, IF=0.246.

Phase 3/2017 Dissemination Communication: I. Neamtu, L. E Nita, A. P Chiriac, A. G Rusu, A. Diaconu, N. Tudorachi, L. Mititelu Tartau, Nanogels preparation for controlled bioactives’ delivery, 2nd Annual Conference and Expo on March 27-28, 2017 Madrid, Spain. L. E Nita, A. P Chiriac, A. Diaconu, M. Bercea, M. Asandulesa, Nanostructures based on poly(aspartic acid) and bovine albumin by self-assembling procedure, 2nd Annual Conference and Expo on March 27-28, 2017 Madrid, Spain – Best poster presentation awarded . L.E. Nita, A.G. Rusu, A. Diaconu, A. P. Chiriac, I. Neamtu, Interpenetrating polymer network hydrogel based on poly(dimethylaminoethyl methacrylate) and a tetraoxaspiro [5.5]undecane derivative: synthesis and some characteristics, 9th International Symposium MMOPS Molecular Mobility and Order in Polymer Systems, June 19 – 23, 2017 St. Petersburg, Russia. A. P. Chiriac, L. E. Nita, A. Diaconu, A. Rusu, I. Neamtu, Smart Gel Structures Based on a New Macromolecular Compound and Low Molecular Mass Gelators, 9th International Symposium MMOPS Molecular Mobility and Order in Polymer Systems, June 19 – 23, 2017 St. Petersburg, Russia. L. E Nita, A.P. Chiriac, M. Bercea, A. Diaconu, The self-assembly between amphiphilic natural polymers and synthetic macromolecular compounds into nanostructured particles, 9th International Symposium MMOPS Molecular Mobility and Order in Polymer Systems, June 19 – 23, 2017 St. Petersburg, Russia. L. E. Nita, A. P. Chiriac, M. Bercea, A. Diaconu, A. G. Rusu, L. Mititelu-Tartau , Smart hybrid gel structures based on hyaluronic acid conjugated with a synthetic copolymer, invited conference at Baltic Conference Series (08 - 11 October 2017), Sweden

Conclusions A new synthetic copolymers based on ITA and U was obtained. Optimal parameters for PITAU synthesis were determined varying the molar ratio of co-monomers. The influence induced by composition on the physico-chemical properties of the synthesized polymeric material was studied; To broaden the applicability of the PITAU copolymer, a series of gel-type polymer networks have been successfully prepared to obtain biocompatible materials with potential applications as drug-controlled delivery systems. From the point of view of potential applications in the pharmaceutical field, the prepared gels have the ability to incorporate and release an active principle under simulated physiological conditions. The release rate was influenced by the compositional nature of the gel, previously determined by the different swelling behaviour of the gel.   The compounds described in this study have the characteristics of a biomaterial that can be used in various medical applications to provide biocompatibility, in particular in the preparation of dressings for the treatment of dermal diseases, for the administration of transdermal drugs or as injectable gels for the treatment of diseases of the type arthritis.