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Water-soluble guanidinylated chitosan: a candidate material for protein delivery systems

Polymer Journal volume 55, pages 885–895 (2023)Cite this article

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Abstract

Here, we introduce water-soluble guanidinylated chitosan (WGCS) as a candidate material for protein delivery systems to enhance the cellular internalization of protein/peptide drugs. A WGCS composed of 48.2% guanidinylated chitosan, 20.6% chitosan, and 31.2% chitin units was prepared with a low-molecular-weight chitosan (CS) lactate via a guanidinylation reaction with 1-amidinopyrazole hydrochloride. The Mn of WGCS was estimated by gel permeation chromatography analysis to be 7.6 × 103 (Mw/Mn = 1.5). The higher chitin content in WGCS than in common CS (<20%) is an important factor in achieving water solubility. WGCS showed ca. 2.5-fold higher internalization into HeLa cells than CS does. This clearly indicated that guanidinylation enhances internalization. In addition, endocytic pathways were suggested as a mechanism underlying internalization. Moreover, WGCS significantly enhanced the internalization of bovine serum albumin (BSA) in transport medium at pH 7.4 containing BSA: the internalized amount of BSA in the presence of WGCS was ca. 2-fold higher than in the presence of CS. This higher internalization was caused by efficient binding between WGCS and BSA via electrostatic interactions owing to the guanidino groups. Indeed, the affinity of the binding sites of WGCS is more than 10-fold higher than that of the binding sites of CS.

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References

  1. Ninomiya K, Okura M. Nationwide comprehensive epidemiological study of rare diseases in Japan using a health insurance claims database. Orphanet J Rare Dis. 2022;17:140 https://doi.org/10.1186/s13023-022-02290-0.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 2001;46:3–26. https://doi.org/10.1016/s0169-409x(00)00129-0.

    Article  CAS  PubMed  Google Scholar 

  3. Amidi M, Mastrobattista E, Jiskoot W, Hennink WE. Chitosan-based delivery systems for protein therapeutics and antigens. Adv Drug Deliv Rev. 2010;62:59–82. https://doi.org/10.1016/j.addr.2009.11.009.

    Article  CAS  PubMed  Google Scholar 

  4. Bekale L, Agudelo D, Tajmir-Riahi HA. Effect of polymer molecular weight on chitosan-protein interaction. Colloids Surf B. 2015;125:309–17. https://doi.org/10.1016/j.colsurfb.2014.11.037.

    Article  CAS  Google Scholar 

  5. Bizeau J, Mertz D. Design and applications of protein delivery systems in nanomedicine and tissue engineering. Adv Colloid Interface Sci. 2021;287:102334. https://doi.org/10.1016/j.cis.2020.102334.

    Article  CAS  PubMed  Google Scholar 

  6. Horn JM, Obermeyer AC. Genetic and covalent protein modification strategies to facilitate intracellular delivery. Biomacromolecules. 2021;22:4883–904. https://doi.org/10.1021/acs.biomac.1c00745.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Verma S, Goand UK, Husain A, Katekar RA, Garg R, Gayen JR. Challenges of peptide and protein drug delivery by oral route: current strategies to improve the bioavailability. Drug Dev Res. 2021;82:927–44. https://doi.org/10.1002/ddr.21832.

    Article  CAS  PubMed  Google Scholar 

  8. Zhang R, Nie T, Fang Y, Huang H, Wu J. Poly(disulfide)s: from synthesis to drug delivery. Biomacromolecules. 2022;23:1–19. https://doi.org/10.1021/acs.biomac.1c01210.

    Article  PubMed  Google Scholar 

  9. Izawa H, Haraya YT, Kawakami K. Cyclodextrin-grafted chitosans for pharmaceutical applications. Trends Glycosci Glycotechnol. 2017;29:E93–8.

    Article  Google Scholar 

  10. Kumar MN, Muzzarelli RA, Muzzarelli C, Sashiwa H, Domb AJ. Chitosan chemistry and pharmaceutical perspectives. Chem Rev. 2004;104:6017–84. https://doi.org/10.1021/cr030441b.

    Article  PubMed  Google Scholar 

  11. Izawa H. Preparation of biobased wrinkled surfaces via lignification-mimetic reactions and drying: a new approach for developing surface wrinkling. Polym J. 2017;49:759–65. https://doi.org/10.1038/pj.2017.52.

    Article  CAS  Google Scholar 

  12. Li B, Wang J, Moustafa ME, Yang H. Ecofriendly method to dissolve chitosan in plain water. ACS Biomater Sci Eng. 2019;5:6355–60. https://doi.org/10.1021/acsbiomaterials.9b00695.

    Article  CAS  PubMed  Google Scholar 

  13. Aranaz I, Alcantara AR, Civera MC, Arias C, Elorza B, Heras CA, et al. Chitosan: an overview of its properties and applications. Polymers. 2021;13. https://doi.org/10.3390/polym13193256.

  14. Lee M, Nah JW, Kwon Y, Koh JJ, Ko KS, Kim SW, et al. Water-soluble and low molecular weight chitosan-based plasmid DNA delivery. Pharm Res. 2001;18:427–31. https://doi.org/10.1023/a:1011037807261.

    Article  CAS  PubMed  Google Scholar 

  15. Nakamichi A, Kadokawa J. Fabrication of Chitosan-based network polysaccharide nanogels. Molecules. 2022;27:8384.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kubota N, Eguchi Y. Facile preparation of water-soluble N-acetylated chitosan and molecular weight dependence of its water-solubility. Polym J. 1997;29:123–7. https://doi.org/10.1295/polymj.29.123.

    Article  CAS  Google Scholar 

  17. Suzuki S, Shimahashi K, Takahara J, Sunako M, Takaha T, Ogawa K, et al. Effect of addition of water-soluble chitin on amylose film. Biomacromolecules. 2005;6:3238–42. https://doi.org/10.1021/bm050486h.

    Article  CAS  PubMed  Google Scholar 

  18. Cho YW, Jang J, Park CR, Ko SW. Preparation and solubility in acid and water of partially deacetylated chitins. Biomacromolecules. 2000;1:609–14. https://doi.org/10.1021/bm000036j.

    Article  CAS  PubMed  Google Scholar 

  19. Gorochovceva N, Makuška R. Synthesis and study of water-soluble chitosan-O-poly(ethylene glycol) graft copolymers. Eur Polym J. 2004;40:685–91. https://doi.org/10.1016/j.eurpolymj.2003.12.005.

    Article  CAS  Google Scholar 

  20. Mao S, Shuai X, Unger F, Wittmar M, Xie X, Kissel T. Synthesis, characterization and cytotoxicity of poly(ethylene glycol)-graft-trimethyl chitosan block copolymers. Biomaterials. 2005;26:6343–56. https://doi.org/10.1016/j.biomaterials.2005.03.036.

    Article  CAS  PubMed  Google Scholar 

  21. Kumar S, Dutta J, Dutta PK, Koh J. A systematic study on chitosan-liposome based systems for biomedical applications. Int J Biol Macromol. 2020;160:470–81. https://doi.org/10.1016/j.ijbiomac.2020.05.192.

    Article  CAS  PubMed  Google Scholar 

  22. Lombardo R, Musumeci T, Carbone C, Pignatello R. Nanotechnologies for intranasal drug delivery: an update of literature. Pharm Dev Technol. 2021;26:824–45. https://doi.org/10.1080/10837450.2021.1950186.

    Article  CAS  PubMed  Google Scholar 

  23. George M, Abraham TE. Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan-a review. J Control Release. 2006;114:1–14. https://doi.org/10.1016/j.jconrel.2006.04.017.

    Article  CAS  PubMed  Google Scholar 

  24. Izawa H, Kinai M, Ifuku S, Morimoto M, Saimoto H. Guanidinylated chitosan inspired by arginine-rich cell-penetrating peptides. Int J Biol Macromol. 2019;125:901–5.

    Article  CAS  PubMed  Google Scholar 

  25. Izawa, H, Kinai, M, Ifuku, S, Morimoto, M & Saimoto, H. Guanidinylation of chitooligosaccharides involving internal cyclization of the guanidino group on the reducing end and effect of guanidinylation on protein binding ability. Biomolecules. 2019;9. https://doi.org/10.3390/biom9070259.

  26. Mogaki R, Hashim PK, Okuro K, Aida T. Guanidinium-based “molecular glues” for modulation of biomolecular functions. Chem Soc Rev. 2017;46:6480–91. https://doi.org/10.1039/c7cs00647k.

    Article  CAS  PubMed  Google Scholar 

  27. Khafagy ES, Morishita M. Oral biodrug delivery using cell-penetrating peptide. Adv Drug Deliv Rev. 2012;64:531–9.

    Article  CAS  PubMed  Google Scholar 

  28. Kristensen M, Nielsen HM. Cell-penetrating peptides as carriers for oral delivery of biopharmaceuticals. Basic Clin Pharm Toxicol. 2016;118:99–106. https://doi.org/10.1111/bcpt.12515.

    Article  CAS  Google Scholar 

  29. Murayama T, Masuda T, Afonin S, Kawano K, Takatani-Nakase T, Ida H, et al. Loosening of lipid packing promotes oligoarginine entry into cells. Angew Chem Int Ed. 2017;56:7644–7. https://doi.org/10.1002/anie.201703578.

    Article  CAS  Google Scholar 

  30. Takeuchi T, Futaki S. Current understanding of direct translocation of arginine-rich cell-penetrating peptides and its internalization mechanisms. Chem Pharm Bull. 2016;64:1431–7. https://doi.org/10.1248/cpb.c16-00505.

    Article  CAS  Google Scholar 

  31. Hu Y, Du YM, Yang JH, Kennedy JF, Wang XH, Wang LS. Synthesis, characterization and antibacterial activity of guanidinylated chitosan. Carbohydr Polym. 2007;67:66–72. https://doi.org/10.1016/j.carbpol.2006.04.015.

    Article  CAS  Google Scholar 

  32. Sahariah P, Oskarsson BM, Hjalmarsdottir MA, Masson M. Synthesis of guanidinylated chitosan with the aid of multiple protecting groups and investigation of antibacterial activity. Carbohydr Polym. 2015;127:407–17. https://doi.org/10.1016/j.carbpol.2015.03.061.

    Article  CAS  PubMed  Google Scholar 

  33. Salama A, Hasanin M, Hesemann P. Synthesis and antimicrobial properties of new chitosan derivatives containing guanidinium groups. Carbohydr Polym. 2020;241:116363. https://doi.org/10.1016/j.carbpol.2020.116363.

    Article  CAS  PubMed  Google Scholar 

  34. Zhang X, Fan J, Lee C, Kim S, Chen C, Lee M. Supramolecular hydrogels based on nanoclay and guanidine-rich chitosan: injectable and moldable osteoinductive carriers. ACS Appl Mater Interfaces. 2020;12:16088–96. https://doi.org/10.1021/acsami.0c01241.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yano K, Masaoka Y, Kataoka M, Sakuma S, Yamashita S. Mechanisms of membrane transport of poorly soluble drugs: role of micelles in oral absorption processes. J Pharm Sci. 2010;99:1336–45. https://doi.org/10.1002/jps.21919.

    Article  CAS  PubMed  Google Scholar 

  36. Shi H, He X, Yuan Y, Wang K, Liu D. Nanoparticle-based biocompatible and long-life marker for lysosome labeling and tracking. Anal Chem. 2010;82:2213–20. https://doi.org/10.1021/ac902417s.

    Article  CAS  PubMed  Google Scholar 

  37. Kamei N, Shigei C, Hasegawa R, Takeda-Morishita M. Exploration of the key factors for optimizing the in vivo oral delivery of insulin by using a noncovalent strategy with cell-penetrating peptides. Biol Pharm Bull. 2018;41:239–46.

    Article  CAS  PubMed  Google Scholar 

  38. Uusna J, Langel K, Langel U. Toxicity, immunogenicity, uptake, and kinetics methods for CPPs. Methods Mol Biol. 2015;1324:133–48. https://doi.org/10.1007/978-1-4939-2806-4_9.

    Article  PubMed  Google Scholar 

  39. Cavanagh RJ, Smith PA, Stolnik S. Exposure to a nonionic surfactant induces a response akin to heat-shock apoptosis in intestinal epithelial cells: implications for excipients safety. Mol Pharm. 2019;16:618–31. https://doi.org/10.1021/acs.molpharmaceut.8b00934.

    Article  CAS  PubMed  Google Scholar 

  40. Muller-Esparza H, Osorio-Valeriano M, Steube N, Thanbichler M, Randau L. Bio-layer interferometry analysis of the target binding activity of CRISPR-cas effector complexes. Front Mol Biosci. 2020;7:98. https://doi.org/10.3389/fmolb.2020.00098.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Fivash M, Towler EM, Fisher RJ. BIAcore for macromolecular interaction. Curr Opin Biotechnol. 1998;9:97–101. https://doi.org/10.1016/s0958-1669(98)80091-8.

    Article  CAS  PubMed  Google Scholar 

  42. Du JR, Su X, Feng X. Chitosan/sericin blend membranes for adsorption of bovine serum albumin. Can J Chem Eng. 2017;95:954–60. https://doi.org/10.1002/cjce.22760.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank Prof. Hiroyuki Saimoto (Tottori University) for valuable discussions. This work was supported in part by JSPS KAKENHI Grant Number 19K05616.

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Authors and Affiliations

  1. Faculty of Engineering, University of Miyazaki, 1-1 Gakuen Kibanadai-Nishi, Miyazaki, 889-2192, Japan

    Hironori Izawa

  2. Department of Chemistry and Biotechnology, Faculty of Engineering, Tottori University, 4-101 Koyama-Minami, Tottori, 680-8550, Japan

    Hironori Izawa & Shinsuke Ifuku

  3. Center for Research on Green Sustainable Chemistry, Tottori University, Tottori, 680-8550, Japan

    Hironori Izawa & Shinsuke Ifuku

  4. Graduate School of Sustainability Science, Tottori University, 4-101 Koyama-Minami, Tottori, 680-8550, Japan

    Ayaka Yagi & Ryo Umemoto

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Correspondence to Hironori Izawa.

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Izawa, H., Yagi, A., Umemoto, R. et al. Water-soluble guanidinylated chitosan: a candidate material for protein delivery systems. Polym J 55, 885–895 (2023). https://doi.org/10.1038/s41428-023-00787-4

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