Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Nanostructure of hyaluronan acyl-derivatives in the solid state Josef Chmelař a, ⁎ , Petr Bělský b , Jiří Mrázek a , Daniel Švadlák a , Martina Hermannová a , Miroslav Šlouf c , Ivan Krakovský d , Daniela Šmejkalová a , Vladimír Velebný a a Contipro a.s., Dolní Dobrouč 401, 561 02, Czech Republic b New Technologies Research Centre, University of West Bohemia, Univerzitní 8, 306 14 Pilsen, Czech Republic c Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského náměstí 2, 162 06 Prague 6, Czech Republic d Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holešovičkách 2, 180 00 Prague 8, Czech Republic ARTICLE INFO Keywords: Hyaluronan Hydrophobization Nanostructure Solid-state Small-angle X-ray scattering Electron microscopy ABSTRACT Acyl derivatives of hyaluronan (acyl-HA) are promising materials for biomedical applications. Depending on the acyl length and the degree of substitution, these derivatives range from self-assembling water-soluble polymers to materials insoluble in aqueous environments. The behaviour of acyl-HA was studied in solution, but little attention was paid to the solid state, despite its importance for applications such as medical device fabrication. We thus used X-ray scattering and electron microscopy to explore the solid-state nano-structure of acyl-HA. The set of samples included various substituents, substitution degrees and molecular weights. The obtained data showed that all studied acyl-HA materials contained structures with dimensions on the order of nanometres that were not present in unmodified HA. The size of the nanostructures increased with the acyl length, while the degree of substitution and molecular weight had negligible effects. We suggest that the observed nanostructure corresponds to a distribution of hydrophobic domains in a hydrophilic matrix of unmodified HA segments. 1. Introduction Hyaluronan (HA) is a naturally occurring anionic linear poly- saccharide with the repeating unit consisting of D-glucuronic acid and N-acetyl-D-glucosamine linked by alternating β(1 → 4) and β(1 → 3) glycosidic bonds. HA is present, for example, in the extracellular ma- trix, skin, synovial fluid, vitreous humour, or various connective tissues. It plays an important role in processes such as tissue hydration, lu- brication, or wound healing (Dicker et al., 2014). As an important constituent of our body, HA is biocompatible, biodegradable and non- toxic (Garg & Hales, 2004), making it a promising material for appli- cations in medicine. Due to its hydrophilic character, native HA is soluble in water. However, insolubility or limited solubility is required for applications such as medical device fabrication or drug delivery. A common ap- proach for overcoming HA solubility is covalent cross-linking. The published methods include processes based on enzymatic reactions (Dvorakova et al., 2014), photochemistry (Bobula et al., 2015), or chemical cross-linking agents such as polyvalent hydrazides (Vercruysse, Marecak, Marecek, & Prestwich, 1997), glutaraldehyde, divinyl sulfone and carbodiimides (Collins & Birkinshaw, 2007, 2008). Another option is to chemically modify HA with hydrophobic side groups such as long acyl chains (Creuzet, Kadi, Rinaudo, & Auzély- Velty, 2006; Finelli et al., 2014; Huerta-Angeles, Bobek, Příkopová, Šmejkalová, & Velebný, 2014; Šmejkalová et al., 2012) or octenyl succinic anhydride (Eenschooten et al., 2012). These hydrophobized derivatives can be used in drug delivery applications (Eenschooten et al., 2012; Choi et al., 2010; Šmejkalová et al., 2017) or for the pre- paration of polymeric films (Foglarová et al., 2016) and fibres (Zápotocký et al., 2016). The nanostructure of biomaterials can have a significant impact on their performance in applications (Tang et al., 2016). In the case of native HA, there is a number of studies concerned with the nanos- tructure and chain conformations both in the solid state (Cowman & Matsuoka, 2005; Haxaire, Braccini, Milas, Rinaudo, & Perez, 2000) and in aqueous solutions (Buhler & Boué, 2004; Cowman & Matsuoka, 2005; Matteini et al., 2009). On the contrary, the nano-scale behaviour of hydrophobized HA was only studied in solution with emphasis on drug delivery applications (Eenschooten et al., 2012; Šmejkalová et al., 2014). The solid-state structure of these derivatives was not yet ex- plored, despite the intention to use them also in solid products. Apart from its practical importance, this topic is also interesting in primary research, since the nanostructure of polyelectrolytes such as HA is a complex problem (Svergun & Koch, 2003) that is still not completely understood. Small-angle X-ray scattering (SAXS) is commonly used for studying https://doi.org/10.1016/j.carbpol.2018.04.111 Received 1 March 2018; Received in revised form 23 April 2018; Accepted 27 April 2018 ⁎ Corresponding author. E-mail address: Josef.Chmelar@contipro.com (J. Chmelař). Carbohydrate Polymers 195 (2018) 468–475 Available online 30 April 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved. T