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Project titleSynthetic cryogels activated with oligopeptides for the regeneration of nerve fibers

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-209 - Biotechnology (including biological nanotechnology)

Keywordsneuroregeneration, spinal cord injury, polymeric scaffolds, macroporous cryogels, synthetic peptides, affinity association, viral vectors, growth factor analogs, extracellular matrix analogs

Annotation
The development of effective methods for the regeneration of tissues of the nervous system after injuries remains a worldwide challenge. A promising approach is the use of new generation tissue-replacing materials that support the growth of nerve fibers and overcome the shortcomings of the existing approaches. The latter, in particular, are based on the use of recombinant growth factors and cell products with increased risk of side effects and reduced reproducibility of properties. The proposed project aims to create synthetic analogues of the extracellular matrix (ECM) for efficient nerve regeneration with a focus on spinal cord injury. It will be developed a cryogel scaffold that supports nerve fiber outgrowth owing to its controlled three-dimensional and chemical structure, which can be activated with multifunctional oligopeptides that mimic the properties of ECM proteins and growth factors. The key hypothesis of the project is that the combination of a macroporous cryogel and biomimetic oligopeptides will provide a synergistic regenerative activity in relation to damaged nerve tissues, comparable to the action of highly active biological products. In the course of the project, a number of multicomponent biodegradable cryogels based on PEG-macromer, close in their mechanical properties to the tissues of the spinal cord, will be designed and characterized. Approaches to controlled activation of the cryogels by individual oligopeptides and their compositions through affinity immobilization will be tested. The effects of immobilized oligopeptides on migration, proliferation and functional activity of neuronal cells in a three-dimensional microenvironment will be evaluated. In addition, the possibility of peptide-mediated immobilization of a model adenoviral construct within the cryogels to deliver the reporter gene to target cells will be explored. Although viral preparations have disadvantages typical for recombinant products, it is assumed that the developed technology will allow a significant reduction in the required amount of virus encoding for the therapeutic protein, while achieving high transduction/regeneration efficiency. The results of the proposed exploratory study will contribute to understanding the role of three-dimensionally organized oligopeptide signals in the behavior of neuronal cells and will allow us to evaluate the therapeutic potential of new bioactivated cryogel scaffolds in neuroregeneration.

Expected results
The project aims to create a new tissue engineering technology using a biocompatible and biodegradable macroporous cryogel based on a cationized oligo-polyethylene glycol macromer [OPF] and synthetic oligopeptides that mimic the properties of ECM/growth factors for effective regeneration of damaged nerve fibers. The project includes the design of multifunctional cryogels, the study of the processes of their affinity activation using oligopeptides and adenovirus constructs, as well as an assessment of the neuroregenerative potential of the created scaffolds. Main expected results: 1. Characteristics of the conditions for the formation of macroporous hydrogels (cryogels) based on [OPF] by cryotropic gelation. Data on the effect of cationic monomers (MAETAC, and others) and the receptor component (acryloyl-cyclodextrin) on the structural and viscoelastic properties of [OPF] cryogels (Appendix 2). 2. Results of studying the migration, proliferation, and differentiation of neuronal cells in [OPF] cryogels of various compositions. 3. Formation of [OPF] cryogels in the form of conduits with different geometries to replace spinal cord defects. Results of a comparative assessment of the viscoelastic properties of conduits and explants of the spinal cord of mammals. 4. Solid-phase synthesis of oligopeptides containing an affinity group, biomimetic sequences, and a fluorescent label. Characterization of the processes of affinity immobilization of oligopeptides in [OPF] cryogel. 5. Data on the stimulating/synergistic effect of the oligopeptide components of the [OPF] cryogel on the growth and functional activity of neuronal cells in a three-dimensional environment. Selection of a scaffold for further in vivo studies in a model of spinal cord injury. 6. Design and production of [OPF] cryogel for affinity immobilization of a model adenoviral construct with the GFP gene. The results of the study of the efficiency of viral transduction toward cells of the stromal-vascular fraction cultured in [OPF]-cryogel with different loadings of the viral vector. Assessment of the distribution of the viral vector in the scaffold according to the fluorescence profile of the reporter gene. The results will serve as the basis for the creation of new generation biocompatible and biodegradable synthetic cryogel scaffolds for post-traumatic regeneration of nerve fibers. They will contribute to the development of approaches related to the affinity activation of cryogels by biomimetic oligopeptides, understanding the role of the three-dimensional organization of oligopeptide signals in the functioning of neuronal cells, and will also allow evaluating the potential for peptide-mediated association and delivery of viral preparations to target cells. The implementation of the fundamental and applied tasks of the project will make it possible to propose multifunctional cryogel scaffolds that are of interest for the treatment of traumatic diseases of the central and peripheral nervous systems, overcoming the limitations of existing biomaterials with encapsulated cellular and recombinant products.

Annotation of the results obtained in 2022
Oligo (poly (ethylene glycol) fumarate) (OPF)-based macroporous cryogels were developed as a potential biodegradable scaffold for spinal cord repair. A series of OPF cryogel conduits in combination with PEG diacrylate and 2-(methacryloyloxy) ethyl-trimethylammonium chloride (MAETAC) cationic monomers were synthesized and characterized. The obtained results for the first time propose the OPF-based multicomponent cryogels as potential biomaterials with multiple benefits for neural tissue regeneration. Important relationships between the composition, viscoelastic properties, content of capillary and polymer-bound water, and porous structure of the cryogels were established and the contributions of each component to these properties were revealed. The viscoelastic properties were comprehensively compared to those previously reported for neural tissues and scaffolds in order to identify working ranges of the elastic modulus of implantable cryogels. The ability of the cryogels to effectively support adhesion, migration, and proliferation of neuronal cells was demonstrated. The obtained results in the course of the project show that the OPF-based cryogels can be recognized as a biomaterial platform to produce spinal cord-replacing conduits with tunable properties and amenability to further modification with neuroactive factors. Additionally, the neuronal cell-modulating potentials of transition metal microelements incorporated in peptide amphiphile-based nanofibers were for the first time revealed, suggesting transition metals as promising agents for the activation/modification of peptide-based biomaterials. The effects of Zn, Cu, and Mn ions were assessed in relation to their prooxidant potentials and the TM-activated PA scaffolds were proven supportive of cell adhesion, viability and growth of PC-12 neuronal cells. The results highlight a particular role of Mn (II) as a neuromodulator agent in cell-matrix interaction and induction of neuritogenesis process.

Publications

1. Dayob K., Zengin A., Garifullin R., Guler M.O., Abdullin T.I., Yergeshov A., Salakhieva D.V., Cong H.H., Zoughaib M. Metal-Chelating Self-Assembling Peptide Nanofiber Scaffolds for Modulation of Neuronal Cell Behavior Micromachines, vol. 14, issue 4, page 883. (year - 2023) https://doi.org/10.3390/mi14040883

2. Zoughaib M., Dayob K., Avdokushina S., Kamalov M.I., Salakhieva D.V., Savina I.N., Lavrov I.A., Abdullin T.I. Oligo (Poly (Ethylene Glycol) Fumarate)-Based Multicomponent Cryogels for Neural Tissue Replacement Gels, vol. 9, issue 2, page 105. (year - 2023) https://doi.org/10.3390/gels9020105

3. Avdokushina S.M., Dayob K., Kamalov M.I., Abdullin T.I., Zoughaib M. ХАРАКТЕРИСТИКА СИНТЕТИЧЕСКОГО ГИДРОГЕЛЯ В КАЧЕСТВЕ МАТРИКСА ДЛЯ НЕЙРОНАЛЬНЫХ КЛЕТОК Казанский (Приволжский) федеральный университет (МАТЕРИАЛЫ И ТЕХНОЛОГИИ XXI ВЕКА), V МАТЕРИАЛЫ И ТЕХНОЛОГИИ XXI ВЕКА, 2022, СБОРНИК ТЕЗИСОВ, c. 7 (year - 2022)

4. Avdokushina S.M., Kamalov M.I., Abdullin T.I., Lavrov I.A., Zoughaib M. ПОЛУЧЕНИЕ БИОДЕГРАДИРУЕМЫХ ГИДРОГЕЛЕЙ НА ОСНОВЕ ПЭГ И ИССЛЕДОВАНИЕ ИХ СОВМЕСТИМОСТИ С НЕЙРОНАЛЬНЫМИ КЛЕТКАМИ Гены и клетки, Гены и клетки, Том XVII, №3, C. 8, 2022 (year - 2022)

5. Zoughaib M., Abdullin T.I. SYNTHETIC OLIGOPEPTIDE-MODIFIED BIOMATERIALS Гены и клетки, Гены и клетки, Том XVII, №3, C. 6, 2022 (year - 2022)