Introduction: Chemical modification of hyaluronic acid (HA) is a key aspect for the production of advanced naturally derived biomaterials. The grafting of hydrophobic moieties to HA is of particular interest to generate amphiphilic species that can self-assemble in supramolecular structures[1]. In this study, we prepared and characterized a series of short alkyl derivatives of HA with a broad range of substitution degree (DS). A novel derivatization method based on amidation promoted by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) was used[2]. Remarkable rheological properties were observed.
Materials and Methods: HA-butylamine (HA-B) and HA-propylamine (HA-P) were synthesized by reacting HA (molecular weight = 280 kDa) with the amines in presence of DMTMM in MES buffer, pH = 5.5, at different temperatures, molar ratios between reagents and multiple feeding. The molar degree of substitution (DS) was determined via 1H NMR (Bruker Avance AV-500 18 MHz spectrometer). Flow and oscillatory rheological measurements were performed for 5% v/w solutions of HA-B and HA-P with an Anton Paar MCR-302 rheometer. As some derivatives were non-soluble, a thermal treatment (92°C for 1h 30', sufficient to make them soluble) was applied to all specimens before characterization. Absence of free amine groups in the final product was assessed via Ninhydrin assay.
Results: Synthesis. At higher temperature, DMTMM was deactivated faster. Also the coupling proceeded faster, with overall yield increase (fig 1). DS of up to 51% was achieved with multiple amine additions.
Rheological properties. Storage modulus G' increased with the increase of DS up to a maximum at DS = 3.7% (HA-B) and 3% (HA-P), and decreased for higher DS (fig 2). Similar behavior was observed for the viscosity curve (not shown), with maximum zero-shear viscosity and shear-thinning gradient for the DS above indicated.
Fig. 1: Kinetics of HA-P formation at 56°C and at room temperature (RT), also upon multiple amine additions (empty squares).
Fig. 2: G' at RT versus DS (ratio HA:DMTMM: amine 1:1:0.5).
Discussion: A broad range of HA modifications with grafted short alkyl groups was achieved. Upon changing the DS values, remarkable changes in the rheological properties were observed. The complex dependence of G' on DS suggests a structural rearrangement with associative behaviour maximized at DS= 3.7% (HA-B) and 3% (HA-P). The non-solubility of most as-prepared derivatives before thermal treatment supports this hypothesis. At the moment, deeper structural analyses are ongoing.
Conclusion: The DMTMM grafting strategy introduced here is a viable method for fine-tuning the rheological properties of HA with minimal modification of the polymer. Synthesis conditions were optimized, resulting in superior efficiency at high temperature despite faster DMTMM deactivation. Owing to their singular rheological profile, HA-B and HA-P are valuable biomaterial candidates for preparing bio-inks or hydrogels for drug delivery and regenerative medicine.
Ms. Doris Sutter from ETH-Zurich, for performing NMR measurements
References:
[1] C. Eenschooten et al. (2012) Carbohydr Polym 87:444-451
[2] M. D’Este, D. Eglin, M. Alini (2014) Carbohydr Polym 108:239-46