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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
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.
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.
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.
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.
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.
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.
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.
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.
Izawa H, Haraya YT, Kawakami K. Cyclodextrin-grafted chitosans for pharmaceutical applications. Trends Glycosci Glycotechnol. 2017;29:E93–8.
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.
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.
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.
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.
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.
Nakamichi A, Kadokawa J. Fabrication of Chitosan-based network polysaccharide nanogels. Molecules. 2022;27:8384.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Khafagy ES, Morishita M. Oral biodrug delivery using cell-penetrating peptide. Adv Drug Deliv Rev. 2012;64:531–9.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Acknowledgements
The authors thank Prof. Hiroyuki Saimoto (Tottori University) for valuable discussions. This work was supported in part by JSPS KAKENHI Grant Number 19K05616.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41428-023-00787-4