Ukr.Biochem.J. 2024; Volume 96, Issue 1, Jan-Feb, pp. 80-95


Multifunctional chitosan-based hydrogels: characterization and evaluation of biocompatibility and biodegradability in vitro

N. Manko1, M. Lootsik1, V. Antonyuk1,2, I. Ivasechko1,
N. Skorokhyd1, H. Kosiakova3, O. Mehed’3, T. Horid’ko3,
N. Hula3, O. Klyuchivska1, R. Panchuk1, N. Pokhodylo4,
О. Barabash4, T. Dumych2, R. Stoika1,4*

1Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv;
2Danylo Halytsky National Medical University of Lviv, Lviv, Ukraine;
3Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
4Ivan Franko National University of Lviv, Lviv, Ukraine;

Received: 29 November 2023; Revised: 31 December 2023;
Accepted: 01 February 2024; Available on-line: 26 February 2024

Creation of novel remedies efficient in supporting wound healing remains an actual task in pharmacology. Hydrogels showed high efficiency in wound healing and tissue regeneration due to viscosity, elasticity and fluidity that provide them with functional characteristics similar to that in extracellular matrix. The aim of the study was to create chitosan-based hydrogels functionalized with different components (chondroitin-6-sulfate, hyaluronic acid, N-stearoylethanolamine) and to estimate their biocompatibility and biodegradabili­ty in vitro. For the first time, a lipid substance N-stearoylethanolamine (NSE) known as suppressor of pro-inflammatory cytokines expression was used as hydrogel component (1.95 mg/g). FTIR analysis confirmed the complexation of chitosan molecule with hyaluronate, chondroitin-6-sulfate, NSE. MTT-test and Trypan blue exclusion test were used to study hydrogels cytotoxicity towards human cells of different tissue origin. Biodegradability of hydrogels was evaluated using direct hydrogel contact with cells and cell-independent degradation. It was shown that chondroitin-6-sulfate (<2 mg/ml), hyaluronic acid (<2 mg/ml) and NSE (26 µg/ml) did not demonstrate significant toxic effects towards pseudonormal human cells of the MCF10A, HaCat, HEK293 lines and mouse cells of the Balb/3T3 line. The studied hydrogels were stable in saline solution, while in a complete culture medium containing 10% fetal bovine blood serum they underwent degradation in >24 h. The identified biodegradability of the chitosan-based hydrogels is important for the release of noncovalently immobilized NSE into biological medium. Further studies on laboratory animals with experimental wounds are expected to explore the potential of created hydrogels as anti-inflammatory and wound-healing agents.

Keywords: , , , , , , ,


  1. Dunn L, Prosser HC, Tan JT, Vanags LZ, Ng MK, Bursill CA. Murine model of wound healing. J Vis Exp. 2013;(75):e50265. PubMed, PubMedCentral, CrossRef
  2. Masson-Meyers DS, Andrade TAM, Caetano GF, Guimaraes FR, Leite MN, Leite SN, Frade MAC. Experimental models and methods for cutaneous wound healing assessment. Int J Exp Pathol. 2020;101(1-2):21-37. PubMed, PubMedCentral, CrossRef
  3. Nuutila K, Eriksson E. Moist Wound Healing with Commonly Available Dressings. Adv Wound Care (New Rochelle). 2021;10(12):685-698. PubMed, PubMedCentral, CrossRef
  4. Feng P, Luo Y, Ke C, Qiu H, Wang W, Zhu Y, Hou R, Xu L, Wu S. Chitosan-Based Functional Materials for Skin Wound Repair: Mechanisms and Applications. Front Bioeng Biotechnol. 2021;9:650598. PubMed, PubMedCentral, CrossRef
  5. Alsarra IA. Chitosan topical gel formulation in the management of burn wounds. Int J Biol Macromol. 2009;45(1):16-21 PubMed, CrossRef
  6. Wu G, Ma F, Xue Y, Peng Y, Hu L, Kang X, Sun Q, Ouyang DF, Tang B, Lin L. Chondroitin sulfate zinc with antibacterial properties and anti-inflammatory effects for skin wound healing. Carbohydr Polym. 2022;278:118996. PubMed, CrossRef
  7. Frenkel JS. The role of hyaluronan in wound healing. Int Wound J. 2014;11(2):159-163. PubMed, PubMedCentral, CrossRef
  8. Kosiakova H, Berdyshev A, Dosenko V, Drevytska T, Herasymenko O, Hula N. The involvement of peroxisome proliferator-activated receptor gamma (PPARγ) in anti-inflammatory activity of N-stearoylethanolamine. Heliyon. 2022;8(11):e11336. PubMed, PubMedCentral, CrossRef
  9. Pils V, Terlecki-Zaniewicz L, Schosserer M, Grillari J, Lämmermann I. The role of lipid-based signalling in wound healing and senescence. Mech Ageing Dev. 2021;198:111527. PubMed, CrossRef
  10. Patent of Ukraine for Invention UA N112716 (C08B), Method of preparing chitosan with high hemostatic activity. Аuthors: Lootsik MD, Bilyy RО, Lutsik ММ, Stoika RS. Published in 10.10.2016.
  11. Patent 81861 UA, IPC (2007.01) С07С 215/00, С07С 229/02. Method of preparing N-acylethanolamins. Authors: Hula NM, Маrgytych VМ, Hоrydko ТМ, Аrtаmоnоv МV, Zhukov ОD, Klymashevskyy VМ. Publ. 11.02.2008. Bull. No 3.
  12. Gilli R, Kacuráková M, Mathlouthi M, Navarini L, Paoletti S. FTIR studies of sodium hyaluronate and its oligomers in the amorphous solid phase and in aqueous solution. Carbohydr Res. 1994;263(2):315-326. PubMed, CrossRef
  13. Keereetaweep J, Chapman KD. Lipidomic Analysis of Endocannabinoid Signaling: Targeted Metabolite Identification and Quantification. Neural Plast. 2016;2016:2426398. PubMed, PubMedCentral, CrossRef
  14. Panchuk R, Skorokhyd N, Chumak V, Lehka L, Kosiakova H, Horid’ko T, Hudz I, Hula N, Riabtseva A, Mitina N, Zaichenko A, Heffeter P, Berger W, Stoika R. Cannabimimetic N-Stearoylethanolamine as “Double-Edged Sword” in Anticancer Chemotherapy: Proapoptotic Effect on Tumor Cells and Suppression of Tumor Growth versus Its Bio-Protective Actions in Complex with Polymeric Carrier on General Toxicity of Doxorubicin In Vivo. Pharmaceutics. 2023;15(3):835. PubMed, PubMedCentral, CrossRef
  15. ISO 10993-5:2009 Biological evaluation of medical devices. Part 5: Tests for in vitro cytotoxicity.
  16. Tyliszczak B, Drabczyk A, Kudłacik-Kramarczyk S, Bialik-Wąs K, Sobczak-Kupiec A. In vitro cytotoxicity of hydrogels based on chitosan and modified with gold nanoparticles. J Polym Res. 2017;24:153. CrossRef
  17. Thankam FG, Muthu J. Influence of physical and mechanical properties of amphiphilic biosynthetic hydrogels on long-term cell viability. J Mech Behav Biomed Mater. 2014;35:111-122. PubMed, CrossRef
  18. Albinali KE, Zagho MM, Deng Y, Elzatahry AA. A perspective on magnetic core-shell carriers for responsive and targeted drug delivery systems. Int J Nanomedicine. 2019;14:1707-1723. PubMed, PubMedCentral, CrossRef
  19. Siafaka PI, Üstündağ Okur N, Karavas E, Bikiaris DN. Surface Modified Multifunctional and Stimuli Responsive Nanoparticles for Drug Targeting: Current Status and Uses. Int J Mol Sci. 2016;17(9):1440. PubMed, PubMedCentral, CrossRef
  20. dos Santos KS, Coelho JF, Ferreira P, Pinto I, Lorenzetti SG, Ferreira EI, Higa OZ, Gil MH. Synthesis and characterization of membranes obtained by graft copolymerization of 2-hydroxyethyl methacrylate and acrylic acid onto chitosan. Int J Pharm. 2006;310(1-2):37-45. PubMed, CrossRef
  21. Finotelli PV, Da Silva D, Sola-Penna M, Rossi AM, Farina M, Andrade LR, Takeuchi AY, Rocha-Leão MH. Microcapsules of alginate/chitosan containing magnetic nanoparticles for controlled release of insulin. Colloids Surf B Biointerfaces. 2010;81(1):206-211. PubMed, CrossRef
  22. Saboktakin MR, Tabar NA, Tabatabaie RM, Maharramov A, Ramazanov MA. Intelligent Drug Delivery Systems Based on Modified Chitosan Nanoparticles. Lett Org Chem. 2012;9(1):56-70. CrossRef
  23. Lootsik M, Bilyy R, Lutsyk M, Manko N, Navytka S, Kutsiaba V, Stoika R. Honeybee (Apis mellifera) chitosan: purification, heterogeneity and hemocoagulating activity. Biotechnol Acta. 2016;9(6): 39-49. CrossRef
  24. Lootsik M, Bilyy R, Lutsyk M, Stoika R. Preparation of chitosan with high blood clotting activity and its hemostatic potential assessment. Biotechnol Acta. 2015;8(6):32-40. CrossRef
  25. Lootsik M, Manko N, Gromyko O, Tistechok S, Lutsyk M (Jr.) , Stoika R. Honeybee chitosan-melanin complex: isolation and investigation of antimicrobial activity. Ukr Biochem J. 2020;92(6):143-153. CrossRef
  26. Huang G, Huang H. Hyaluronic acid-based biopharmaceutical delivery and tumor-targeted drug delivery system. J Control Release. 2018;278:122-126. PubMed, CrossRef
  27. Yin T, Liu J, Zhao Z, Zhao Y, Dong L, Yang M, Zhou J, Huo M. Redox sensitive hyaluronic acid‐decorated graphene oxide for photothermally controlled tumor‐cytoplasm‐selective rapid drug delivery. Adv Funct Mater. 2017;27(14):1604620. CrossRef
  28. Abednejad A, Ghaee A, Morais ES, Sharma M, Neves BM, Freire MG, Nourmohammadi J, Mehrizi AA. Polyvinylidene fluoride-Hyaluronic acid wound dressing comprised of ionic liquids for controlled drug delivery and dual therapeutic behavior. Acta Biomater. 2019;100:142-157. PubMed, CrossRef
  29. Huang G, Huang H. Application of hyaluronic acid as carriers in drug delivery. Drug Deliv. 2018;25(1):766-772. PubMed, PubMedCentral, CrossRef
  30. Jana P, Shyam M, Singh S, Jayaprakash V, Dev A. Biodegradable polymers in drug delivery and oral vaccination. Eur Polym J. 2021;142:110155. CrossRef
  31. Vasi AM, Popa MI, Butnaru M, Dodi G, Verestiuc L. Chemical functionalization of hyaluronic acid for drug delivery applications. Mater Sci Eng C Mater Biol Appl. 2014;38:177-185. PubMed, CrossRef
  32. Onopchenko OV, Kosiakova GV, Goridko TM, Berdyschev AG, Meged OF, Hula NM. The effect of N-stearoylethanolamine on the activity of antioxidant enzymes, content of lipid peroxidation products and nitric oxide in the blood plasma and liver of rats with induced insulin-resistance. Ukr Biokhim Zhurn. 2013;85(5):88-96. (In Ukrainian). PubMed, CrossRef
  33. Artamonov MV, Zhukov OD, Horid’ko TM, Klimashevsky VM, Martseniuk OP, Hula NM. Effect of N-stearoylethanolamide and ionizing radiation on lipid components of the liver and heart microsomes in rats. Ukr Biokhim Zhurn. 1999;75(4):81-90. (In Ukrainian). PubMed
  34. Onopchenko OV, Kosiakova GV , Oz M, Klimashevsky VM, Gula NM. N-stearoylethanolamine restores pancreas lipid composition in obesity-induced insulin resistant rats. Lipids. 2015;50(1):13-21. PubMed, CrossRef
  35. Berdyshev AG, Kosiakova HV, Onopchenko OV, Panchuk RR, Stoika RS, Hula NM. N-Stearoylethanolamine suppresses the pro-inflammatory cytokines production by inhibition of NF-κB translocation. Prostaglandins Other Lipid Mediat. 2015;121(Pt A):91-96. PubMed, CrossRef
  36. Zhukov OD, Berdyshe AG, Kosiakova GV, Klimashevskiy VM, Gorid’ko TM, Meged OF, Hula NM. N-stearoylethanolamine effect on the level of 11-hydroxycorticosteroids, cytokines IL-1β, IL-6 and TNFα in rats with nonspecific inflammation caused by thermal burn of skin. Ukr Biochem J. 2014;86(3):88-97. (In Ukrainian). PubMed, CrossRef

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License.