International Journal of Pharmaceutics 420 (2011) 84–92 Contents lists available at SciVerse ScienceDirect International Journal of Pharmaceutics jou rn al h om epa ge: www.elsevier.com/locate/ijpharm Evaluation of the physical and biological properties of hyaluronan and hyaluronan fragments Elaine L. Ferguson a,b,∗ , Jessica L. Roberts a,b , Ryan Moseley a,b , Peter C. Griffiths b,c , David W. Thomas a,b a Wound Biology Group, Tissue Engineering and Reparative Dentistry, School of Dentistry, Cardiff University, Heath Park, Cardiff, CF14 4XY, UK b Cardiff Institute of Tissue Engineering and Repair (CITER), Cardiff University, Cardiff, CF14 4XY, UK c School of Chemistry, Cardiff University, Main Building, Park Place, CF10 3AT, UK a r t i c l e i n f o Article history: Received 15 June 2011 Received in revised form 10 August 2011 Accepted 15 August 2011 Available online 22 August 2011 Keywords: Hyaluronan Wound healing Polymer therapeutics Hyaluronidase a b s t r a c t Hyaluronan (HA) has been extensively used for various medical applications, including osteoarthritis, tissue augmentation and ocular surgery. More recently, it has been investigated for use in polymer therapeutics as a carrier for drugs and biologically active proteins, thanks to its biodegradability, biocom- patibility and inherent biological properties. Such biological functions are strongly dependent on HA’s chain length, yet the molecular weight of HAs used in polymer conjugates varies widely and is inconsis- tent with its intended application. Therefore, this study aimed to determine the ideal chain length of HA to be used in polymer conjugates for enhanced tissue repair. HA fragments (M w 45,000–900,000 g/mol) were prepared by acid hydrolysis of rooster comb HA and their physicochemical and biological properties were characterized. Such HA fragments had a highly extended, almost rod-like solution conformation and demonstrated chain length- and concentration- dependent viscosity, while exposure to HAase caused a rapid reduction in HA viscosity, which was most significant for the native HA. Initial HA hydrolysis rate by HAase varied strongly with HA chain length and was dependent on the formation of a stable enzyme–substrate complex. When normal human dermal fibroblasts were exposed to the different HA fragments for 72 h, only native (900,000 g/mol) HA reduced proliferation at 1000 g/mL. Conversely, only the smallest HA fragment (70,000 g/mol) reduced the pro- liferation of chronic wound fibroblasts, at 1000 g/mL. The 70,000 g/mol HA fragment also promoted the greatest cell attachment. These observations demonstrate that low molecular weight (70,000–120,000 g/mol) HA fragments would be best suited for the delivery of proteins and peptides with applications in chronic wound healing and paves the way for the rationalized development of novel HA conjugates. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Although considerable progress has been made in the field of regenerative medicine, many chronic conditions still prove diffi- cult to treat effectively, due to the complex mechanisms of repair (Herrick et al., 1992). The use of water-soluble polymers in ‘poly- mer therapeutics’ was first described by Duncan (2003), and have been extensively investigated as anticancer nanomedicines. More recently, polymer conjugates, using the biologically inert dex- trin, have been developed to promote tissue repair (Hardwicke et al., 2008, 2010, 2011). We have also demonstrated that hyaluro- nan (HA), a biologically active biopolymer, can be conjugated to trypsin and epidermal growth factor (EGF) to increase resistance to ∗ Corresponding author at: Wound Biology Group, Tissue Engineering and Repar- ative Dentistry, School of Dentistry, Cardiff University, Heath Park, Cardiff, CF14 4XY, UK. Tel.: +44 0 2920 745454; fax: +44 0 29 20742442. E-mail address: FergusonEL@cf.ac.uk (E.L. Ferguson). proteolytic degradation (Ferguson et al., 2010). To date, however, the molecular weight of HA used in polymer conjugates has varied widely and is inconsistent with the intended therapeutic indication (summarized in Table 1). Therefore, this study sought to determine the optimum molecular weight of HA for use as a component of polymer–protein conjugates for tissue repair purposes. HA is a naturally occurring linear polysaccharide that is biodegradable, biocompatible and non-toxic. It is a macromolecule of several million Daltons, consisting of repeating units of d- N-acetylglucosamine and d-glucuronic acid (Linker and Mayer, 1954) that has been widely investigated for its tissue repara- tive effects, due to its multiple potential functions in normal wound healing responses. While some polymers are biologically inert, HA has a crucial role in tissue repair (Itano, 2008). HA dis- plays antioxidant properties, and can modulate wound healing by promoting cell migration and proliferation, facilitating white blood cell infiltration and improving tissue hydration (Chen and Abatangelo, 1999; Price et al., 2007). However, these effects depend, in part, on the molecular size of HA (Huang et al., 2009; Stern 0378-5173/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2011.08.031