Stage 1 (2020): Synthesis of polyssacharide-proteins conjugates and formation of hydrogels. Preliminary studies

A review of the most recent approaches in the field of hydrogels immobilization onto the surface of polymeric film was firstly performed, as the Activity 1.1 of the BioHydroSurf project. Optimum synthesis parameters were selected based on the physico-chemical characterization of the starting components. Moreover, synthesis of chitosan-gelatin and chitosan-fibrinogen was started, evaluating the effect of solution pH on the bioconjugation process. Both the scientific objectives and the results indicators (project web page and participation to one conference) were successfully achieved.

Stage 2 (2021): Synthesis of polyssacharide-proteins conjugates and formation of hydrogels.

The second stage of the project continues the activities on synthesis of polyssacharide-proteins bioconjugates and preparation of hydrogels. The physic-chemical characterization of bioconjugates was performed by spectroscopic methods (FTIR, RMN, UV-Vis) and the bioconjugation degree was evaluated. Hydrogels were prepared from the chitosan-protein conjugates via crosslinking under the action of a chemical or a biocompatible fotoinitiating agent. The gel fraction, rheological properties and structural changes of hydrogels were evaluated, as well as the factors affecting hydrogel formation. The surface of the poly(lactic acid) substrate was functionalized using cold plasma in nitrogen and air gas, at various gas pressure, exposure time and discharge power. Chemical, compositional and wettability properties of the functionalized PLA surfaces were evaluated to determine the optima experimental conditions for plasma activation which will be further used in the next research stage of the project. Both the scientific objectives and the results indicators (update of the project web page, participation to conferences, publication of scientific papers) were successfully achieved.

Figure 1. The experimental protocol for surface functionalization of the PLA polymeric supports with UV- crosslinked CS/G or CS/F hydrogels.

Stage 3 (2022) - Surface immobilization of chitosan-proteins conjugates hydrogels onto PLA film

Hydrogel coatings were obtained by immersion of PLA films (previously functionalized by plasma treatment) into precursor solutions of bioconjugates, followed by crosslinking under UV irradiation. Surface morphology was observed by SEM while the micro-mechanical stability was determined by AFM. Biocompatibility of PLA supports functionalised with chitosan-fibrinogen and chitosan-gelatine bioconjugates was evaluated on fibroblast cultures, observing cell morphology and the cytotoxicity by XTT test and evaluating the amount of adenylate kinase released in culture medium. The functionalized PLA supports maintain adhesion, multiplication and proliferation of fibroblast cells, without affecting cell morphology; this indicates the biocompatibility of polymeric materials.

Figure 2. SEM micrographs of PLA films functionalised at the surface with chitosan-gelatine (CS-G) and chitosan-fibrinogen (CS-FB) bioconjugates UV crosslinked.

The most significant result of the BioHydroSurf project is represented by the synthesis of support platforms for cellular growth, with tridimensionally structured surfaces (of hydrogel type), using as support a biodegradable polymer and an integrated method for functionalization/structuration of surfaces.

Figure 3. Optical microscopy (10X) images of the fibroblast cultures seeded onto PLA/N2 supports functionalized with CS-FB and CS-G bioconjugates, with or without UV crosslinking.

Dissemination

Some of the obtained results in the project BioHydroSurf were presented at 13 scientific conferences (1 conference, 9 oral comunications, 3 posters), were published 5 ISI articles and 1 BDI article, and also 3 book chapters.

Articles - ISI

1. E. Stoleru, R.P. Dumitriu , G.-L. Ailiesei , C. Yilmaz, M. Brebu, Synthesis of bioactive materials by in situ one-step direct loading of syzygium aromaticum essential oil into chitosan-based hydrogels, 2022, 8, 225.
2. E. Stoleru, C. Vasile, A. Irimia, M. Brebu, Towards a bioactive food packaging: poly(lactic acid) surface functionalized by chitosan coating embedding clove and argan oils, 2021, 26(15), 4500.
3. D. Filip, D. Macocinschi, M.-F. Zaltariov, C. A. Gafitanu, C. G. Tuchilus, A. Bele, B.-I. Ciubotaru, E. Stoleru, A. Bargan, Mucoadhesive and antimicrobial allantoin / cyclodextrins-loaded carbopol gels as scaffolds for regenerative medicine, 2022, 8, 416.
4. R.P. Dumitriu, E. Stoleru, G.R. Mitchell, C. Vasile, M. Brebu, Bioactive electrospun fibers of poly(ε-caprolactone) incorporating α-tocopherol for food packaging applications, 2021, 26(18), 5498.
5. E. Stoleru, M. Brebu, Stabilization techniques of essential oils by incorporation into biodegradable polymeric materials for food packaging, 2021, 26(20), 6307.

Articles -  BDI

1. E. Stoleru, R.P. Dumitriu, M. Brebu, C. Vasile, A. Enache, Development of bioactive polymeric materials by incorporation of essential/vegetal oils into biopolymer matrices. Proceedings 2021, 69, 25. https://doi.org/10.3390/CGPM2020-07197.
 

Book chapters

1. E. Stoleru, Poly(Lactic Acid)-based materials: Food packaging applications and biodegradability evaluation, in Applications of Biodegradable and Bio-Based Polymers for Human Health and a Cleaner Environment, I. Stoica, O. Mukbaniani, N. K. Rawat, A. K. Haghi, Eds., Apple Academic Press (Taylor&Francis Group), 2021.
2. E. Stoleru, A. Irimia, E. Butnaru, Chapter 14: Bio-based bioplastics in active food packaging, in Bioplastics for Sustainable Development, M. Kuddus, Z.M.R. Roohi, Eds., Springer, Gateway East, Singapore, 2021, pp. 347-379; https://doi.org/10.1007/978-981-16-1823-9.
3. E. Butnaru, E. Stoleru, A. Irimia, Chapter 15: Synthetic bioplastics in active food packaging, Bioplastics for Sustainable Development, M. Kuddus, Z.M.R. Roohi, Eds., Springer, Gateway East, Singapore, 2021, pp. 381-398; https://doi.org/10.1007/978-981-16-1823-9.

Conferences

1. E. Stoleru, D. Pamfil, R.P. Dumitriu, M. Brebu, Chitosan-based hydrogels and organogels as bioactive materials, XXXIInd edition of the International Congress of “Apollonia” University of Iasi “By promoting excellence, we prepare the future, 28 februarie - 2 martie 2022, Iasi, Romania.

Oral presentations

1. E. Stoleru, D. Pamfil, R. Dumitriu, G. Ailiesei, M. Brebu, Chitosan-protein bioconjugate hydrogels as biocompatible materials, 12th International Conference on Materials Science & Engineering, BRAMAT 2022, 9 - 12 March 2022, Brasov, Romania.
2. E. Stoleru, R. Dumitriu, D.Pamfil, E. Butnaru, M. Brebu, Functional aminopolysachhride-based hydrogels as bioactive and biocompatible materials, International Conference Materials, Methods & Technologies, 19-22 August 2022 Burgas, Bulgaria.
3. E. Stoleru, M. Brebu, R.P. Dumitriu, A. Irimia, Oil-loaded chitosan coatings towards a bioactive food contact material, International Congress of „Apollonia” University from Iași, Edition XXXI, 1 - 3 March 2021, Iasi, Romania.
4. R. P. Dumitriu, A. Irimia, E. Stoleru, M. Brebu, Electrospun fibrous coatings with antimicrobial/antioxidant properties, International Congress of „Apollonia” University from Iași, Edition XXXI, 1 - 3 March 2021, Iasi, Romania.
5. E. Stoleru, R. P. Dumitriu, C. N. Cheaburu-Yilmaz, M. Brebu, Chitosan-based Hydrogels Incorporating Bioactive Compounds, International Conference on Polymer Science and Composite Materials - Polymer Connect, 5-7 July 2021, virtual.
6. R. P. Dumitriu, M. Brebu, E. Stoleru, Alginate/Lignosulfonate-based Bioactive Systems for Green Synthesis of Ag Nanoparticles, International Conference on Polymer Science and Composite Materials - Polymer Connect, 5-7 July 2021, virtual.
7. E. Stoleru, M. Brebu, R.P. Dumitriu, Influence of bioactive plant extract on the stability of chitosan-based materials,Materials, Methods & Technologies International Conference, 19-22 August 2021, Burgas, Bulgaria.
8. E. Stoleru, R.P. Dumitriu, M.A. Brebu, C. Vasile, A. Enache, Development of bioactive polymeric materials by incorporation of essential/vegetal oils into biopolymer matrices, The First International Conference on “Green” Polymer Materials 2020, session Polymer Bioapplications, Online 5-25 noiembrie 2020, https://cgpm2020.sciforum.net, registering DOI: 10.3390/CGPM2020-07197.

Posters

1. D. Pamfil, R. P. Dumitriu, E. Stoleru, Comparative study of EDC/NHS - and riboflavin induced crosslinking of chitosan/gelatin hydrogels, BRAMAT 2022, 12th International Conference on Materials Science & Engineering, 9 – 12 Martie 2022, Brașov, România.
2. E. Stoleru, M. Belous, M. Brebu, Evaluation of plant bioactive compounds activity and stability by spectroscopic methods, 1st SensorFINT International Conference – Non-destructive spectral sensors advances and future trends, 10-12 May 2022, Izola, Slovenia. 
3. E. Stoleru, M. Brebu, Stimuli Responsive Polymeric-Materials as Sensing Platforms, Sensor Fint 2021 - Smart Spectral Sensors for Agri Food Quality and Process Control, 30 September - 1 October 2021, Porto, Portugal.