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

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 M n of WGCS was estimated by gel permeation chromatography analysis to be 7.6 × 10 3 ( M w /M n = 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.