Poly (ethylene glycol)-interpenetrated genipin-crosslinked chitosan hydrogels for controlled drug delivery

Abstract

Smart hydrogels are of increasing interest for controlled drug delivery as they can be used as drug carriers to deliver cargo biomolecules in response to specific physiological signals at tailored rhythm. In this project, pH-responsive hydrogels containing chitosan, genipin, and poly (ethylene glycol) (PEG) are investigated. Owing to good biocompatibility and pH-sensitivity, chitosan was used as the main polymeric backbone, while genipin was employed as a low-toxic crosslinker to bridge chitosan molecules. To enhance the level of control in hydrogel microarchitecture and achieve reproducible properties, PEG was added to form semi-interpenetrating networks. The aim of this project was to develop and evaluate injectable and degradable chitosan-genipin-PEG hydrogels and the feasibility of using them to control drug delivery. The chitosan-genipin hydrogels, with and without PEG, were synthesised under mild conditions (37oC, 24 h) and in a range of shapes (disc, bead, and film). The hydrogels had dark blue colour and intrinsic fluorescence (580 nm excitation and 630 nm emission), due to oxygen radical-induced polymerisation of genipin, as well as the reaction with amino groups of chitosan. The bead-shaped hydrogels were discrete and spherical with diameters ranging from 1 to 30 μm. The disc-shaped hydrogels (13 mm in diameter and 8 mm in height) had microporous structures with pore diameters ranging from 11 to 57 μm and average cross-sectional porous areas of 40% to 64%. Compared to disc-shaped chitosan-genipin hydrogels, presence of PEG up to 1.9 mM generated the same effect as increasing the genipin content, yielding structures with a smaller pore diameter, a lower swelling degree in pH 2 buffer and a higher elastic modulus. Considering cost effectiveness and scale-up production, reducing genipin content by the addition of PEG is favourable. Importantly, hydrogels containing higher concentration of PEG (2.9 mM and above) showed a sudden increase in the swelling degree accompanied with a decrease in the elastic modulus. The release profiles of two drug molecules (perindopril erbumine and 1-methyl D-tryptophan) with different solubility from disc-shaped hydrogels revealed their swelling-controlled kinetic, which fitted well to the Korsmeyer-Peppas model, indicating a non-Fickian transport mechanism. Cytotoxicity assays of hydrogel films towards 3T3 fibroblasts showed that the cells retained normal adhesive properties and high viability on gels with 3.1 mM and 4.4 mM genipin but not on gels with 1.7 mM genipin, suggesting a strong correlation between hydrogels’ stiffness and cell attachment/growth. Adding PEG enhanced the viability of 3T3 cells cultured on hydrogel films. To facilitate comparison, the inflammatory responses of DC 2.4 dendritic cells, RAW 264.7 ii macrophage cells, and bone marrow-derived macrophages to uncrosslinked chitosan and crosslinked chitosan-genipin hydrogel films/beads were investigated. Despite induced mRNA expression of some cytokines in all treated cell types (especially up to 2435-fold increase in interferon-β gene expression found in hydrogel film-exposed DC 2.4), no increased levels of five inflammatory cytokines were detected, suggesting the hypo-inflammatory properties of chitosan-genipin hydrogels. The biodegradation of hydrogel films upon exposure to lysozyme and the biodegradation of macrogels after subcutaneous injection in mice were monitored efficiently using the intrinsic fluorescence of hydrogels. Results suggest that the in vivo degradation rate depends critically on where the hydrogel is deposited in tissues. The subcutaneous injection of hydrogel beads induced interferon-β gene transcription significantly and no local skin lesion was observed, suggesting a good biocompatibility in vivo. Collectively, the findings presented in this study provide valuable guidance to further develop these biocompatible, biodegradable, and injectable chitosan-genipin hydrogels as autonomous drug delivery systems.