In vitro study of partially hydrolyzed poly(2-ethyl-2-oxazolines) as materials for biomedical applications


Polymers based on 2-oxazoline, such as poly(2-ethyl-2-oxazolines) (PETOx), are considered to be a type of ‘pseudopeptide’ with the ability to form novel biomaterials. The hydrolysis of PETOx was carried out to evaluate its use in biomedical applications. In the present work, PETOx samples with a range of molar masses were prepared by living cationic polymerization. Hydrolysis was carried out at time intervals ranging from 15 to 180 min to prepare copolymers with different amounts of ethylene imine units. 1H NMR spectroscopy was used to identify the structure of the hydrolyzed polymers. The dependence of in vitro cell viability on the degree of hydrolysis was determined using three different model cell lines, namely, mouse embryonic 3T3 fibroblasts, pancreatic βTC3 cells, and mouse lymphoid macrophages P388.D1. It was demonstrated that increasing the degree of hydrolysis decreased cell viability for all cell types. Fibroblast cells displayed the highest tolerance; additionally, the effect of polymer size showed no observable significance. Macrophage cells, immune system representatives, displayed the highest sensitivity to contact with hydrolyzed PETOx. The effect of polymer hydrolysis, polymer concentration and the incubation time on cell viability was experimentally observed. Confocal laser-scanning microscopy provided evidence of cellular uptake of pyrene-labeled (co)polymers.

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  1. 1.

    Ballarín-González B, Howard KA. Polycation-based nanoparticle delivery of RNAi therapeutics: adverse effect and solutions. Adv Drug Deliv Rev. 2012;64:1717–29.

    Article  Google Scholar 

  2. 2.

    Davis ME. Nonviral gene delivery systems. Curr Opin Biotechnol. 2002;13:128–31.

    Article  Google Scholar 

  3. 3.

    Segura T, Shea LD. Materials for nonviral gene delivery. Annu Rev Mater Res. 2001;31:25–46.

    Article  Google Scholar 

  4. 4.

    Calafiore R, Basta G. Clinical applications of microencapsulated islets: actual prospectives on progress and challenges. Adv Drug Deliv Rev. 2014;67–68:84–92.

    Article  Google Scholar 

  5. 5.

    Lacík I. Current status in immunoprotection of transplanted islets: focus on Islet micro-encapsulation. Micro and Nano Syst. 2013;5:168–76.

    Article  Google Scholar 

  6. 6.

    Ponce S, Orive G, Hernandez R, Gascon AR, Pedraz JL, De Haan BJ, Faas MM, Mathieu HJ, de Vos P. Chemistry and the biological response against immunoisolating alginate polycation capsules of different composition. Biomaterials. 2006;27:4831–9.

    Article  Google Scholar 

  7. 7.

    Balan V, Verestiuc L. Strategies to improve chitosan hemocompatibility: a review. Eur Polym J. 2014;53:171–88.

    Article  Google Scholar 

  8. 8.

    Zhong D, Jiao Y, Zhang Y, Zhang W, Li N, Zuo Q. Effects of the gene carrier polyethylene imines on structure and function of blood components. Biomaterials. 2013;34:294–305.

    Article  Google Scholar 

  9. 9.

    Kronek J, Paulovicova E, Paulovicova L, Kronekova Z, Luston J. Biocompatibility and immunocompatibility assessment of poly(2-oxazolines). In Practical applications in Biomedical engineering. Rijeka: InTech; 2012. p. 257–84.

    Google Scholar 

  10. 10.

    Kronek J, Kronekova Z, Luston J, Paulovicova E, Paulovicova L, Mendrek B. In vitro bio-immunological and cytotoxicity studies of poly(2-oxazolines). J Mater Sci Mater Med. 2011;22:1725–34.

    Article  Google Scholar 

  11. 11.

    Hoogenboom R, Fijten MWM, Michael AR, Meier MAR, Schubert US. Living cationic polymerizations utilizing an automated synthesizer: high-throughput synthesis of polyoxazolines. Macromol Rapid Commun. 2003;24:92–7.

    Article  Google Scholar 

  12. 12.

    Hoogenboom R, Fijten MWM, Schubert US. Parallel kinetic investigation of 2-oxazoline polymerizations with different initiators as basis for designed copolymer synthesis. J Polym Sci. 2004;42:1830–40.

    Article  Google Scholar 

  13. 13.

    Kronek J, Luston J, Kronekova Z, Paulovicova E, Farkas P, Petrencikova N, Paulovicova L, Janigova I. Synthesis and bioimmunological efficiency of poly(2-oxazolines) containing a free amino group. J Mater Sci Mater Med. 2010;21:879–86.

    Article  Google Scholar 

  14. 14.

    Dworak A, Trzebicka B, Kowalczuk A, Tsvetanov C, Rangelov S. Polyoxazolines-mechanism of synthesis and solution properties. Polimery. 2014;59:88–94.

    Article  Google Scholar 

  15. 15.

    Luxenhofer R, Sahay G, Schulz A, Alakhova D, Bronich TK, Jordan R, Kabanov AV. Structure-property relationship in cytotoxicity and cell uptake of poly(2-oxazoline) amphiphiles. J Control Rel. 2011;153:73–82.

    Article  Google Scholar 

  16. 16.

    Luxenhofer R, Han Y, Schulz A, Tong J, He Z, Kabanov AV, Jordan R. Poly(2-oxazoline)s as polymer therapeutics. Macromol Rapid Commun. 2012;33:1613–31.

    Article  Google Scholar 

  17. 17.

    Adams N, Schubert US. Poly(2-oxazolines) in Biological and Biomedical Application Contexts. Adv Drug Deliv Rev. 2007;59:1504–20.

    Article  Google Scholar 

  18. 18.

    Uyama H, Kobayashi S. Synthesis of poly(2-oxazoline) macromonomers having a vinyl ester group. Macromolecules. 1991;24:614–5.

    Article  Google Scholar 

  19. 19.

    Celebi O. Synthesis and Characterization of Poly(2-Ethyl-2-Oxazoline) Functional Prepolymers and Block Copolymers. Dissertation thesis. URI: Jan 19 2014.

  20. 20.

    Hoogenboom R. Poly(2-oxazoline)s: a polymer class with numerous potential applications. Angew Chem Int Ed. 2009;48:7978–94.

    Article  Google Scholar 

  21. 21.

    Hoogenboom R, Schlaad H. Bioinspired poly(2-oxazoline)s. Polymers. 2011;3:467–88.

    Article  Google Scholar 

  22. 22.

    Van Kuringen HPC, Lenoir J, Adriens E, Bender J, Geest BGD, Hoogenboom R. Partial hydrolysis of poly(2-ethyl-2-oxazoline) and potential implications for biomedical applications. Macromol Biosci. 2012;12:1114–23.

    Article  Google Scholar 

  23. 23.

    Van Kuringen HPC, De la Rosa VR, Fijten MWM, Heuts JPA, Hoogenboom R. Enhanced selectivity for the hydrolysis of block copoly(2-oxazoline)s in ethanol-water resulting in linear poly(ethylene imine) copolymers. Macromol Rapid Commun. 2012;33:827–32.

    Article  Google Scholar 

  24. 24.

    Lin PC, Sung CY, Hsiue HG. Non-viral pH sensitive Gene carriers based on poly((2-ethyl-2-oxazolines)-co-ethyleneimine)-block-poly(2-ethyl-2-oxazolines)-co-ethyleneimine): A study of gene release behavior. J Med Biol Eng. 2012; 365–372.

  25. 25.

    Englert C, Tauhardt L, Hartlieb M, Kempe K, Gottschaldt M, Schubert US. Linear poly(ethylene imine)-based hydrogels for effective binding and release of DNA. Biomacromolecules. 2014;15:1124–31.

    Article  Google Scholar 

  26. 26.

    Hong S, Leroueil PR, Janus EK, Peters JL, Kober MM, Islam MT, Orr BG, Baker Jr, Banaszak Holl MM. Interaction of polycationic polymers with supported lipid bilayers and cells: nanoscale hole formation and enhanced membrane permeability. Bioconjugate Chem. 2006;17:728–34.

    Article  Google Scholar 

  27. 27.

    Rokstad AM, Strand B, Espevik T, Mollnes TE. Biocompatibility and biotolerability assessment of microspheres using a whole blood model. Micro and Nano Syst. 2013;5:177–85.

    Article  Google Scholar 

  28. 28.

    Sohaebuddin SK, Thevenot PT, Baker D, Easton JW, Tang L. Nanomaterial cytotoxicity is composition, size, and cell type dependent. Particle Fibre Toxicol. 2010;7:22.

    Article  Google Scholar 

  29. 29.

    Sherratt JA, Dallon JC. Theoretical models of wound healing: past successes and future challenges. Comptes Rendus Biol. 2002;325:557–64.

    Article  Google Scholar 

  30. 30.

    Hunt NC, Shelton RM, Henderson DJ, Grover LM. Calcium-alginate hydrogel-encapsulated fibroblasts provide sustained release of vascular endothelial growth factor. Tissue Eng A. 2013;19:905–14.

    Article  Google Scholar 

  31. 31.

    Todaro GJ, Green H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol. 1963;17:299–313.

    Article  Google Scholar 

  32. 32.

    Moore C, Cooper GJS. Co-secretion of amylin and insulin from cultured islet β-cells: modulation by nutrient secretagogues, islet hormones and hypoglycemic agents. Biochem Biophys Res Commun. 1991;179:1–9.

    Article  Google Scholar 

  33. 33.

    Efrat S, Linde S, Kofod H, Spector D, Delannoy M, Grant S, Hanahan D, Baekkeskov S. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc Nat Acad Sci USA. 1988;85:9037–41.

    Article  Google Scholar 

  34. 34.

    Kronek J, Paulovičova E, Paulovičova L, Kronekova Z, Lustoň J. Immunomodulatory efficiency of poly(2-oxazolines). J Mater Sci Mater Med. 2012;23:1457–64.

    Article  Google Scholar 

  35. 35.

    Fernandes JC, Qiu X, Winnik FM, Benderdour M, Zhang X, Dai K, Shi Q. Linear polyethylenimine produced by partial acid hydrolysis of poly(2-ethyl-2-oxazoline) for DNA and siRNA delivery in vitro. Int J Nanomed. 2013;8:4091–102.

    Google Scholar 

  36. 36.

    Howard KA. Delivery of RNA interference therapeutics using polycation-based nanoparticles. Adv Drug Deliv Rev. 2009;61:710–20.

    Article  Google Scholar 

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Authors from the Polymer Institute of the Slovak Academy of Sciences are thankful the Slovak Grant Agency, VEGA, for financial support of Projects No. 2/0151/12 and 2/0163/12. Authors from the Centre of Polymer Systems are thankful for the support of the Operational Program Education for Competitiveness co-funded by the European Social Fund (ESF) and the national budget of the Czech Republic, within the framework of the Advanced Theoretical and Experimental Studies of Polymer Systems (reg. number: CZ.1.07/2.3.00/20.0104) Project, including partial support from the Ministry of Education, Youth and Sports of the Czech Republic (Project No: ME-LH14050).

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Correspondence to Juraj Kronek.

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Shah, R., Kronekova, Z., Zahoranová, A. et al. In vitro study of partially hydrolyzed poly(2-ethyl-2-oxazolines) as materials for biomedical applications. J Mater Sci: Mater Med 26, 157 (2015).

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  • High Performance Liquid Chromatography
  • Molar Mass
  • Lower Critical Solution Temperature
  • Cationic Polymer
  • Cationic Polymerization