ARTICLE pubs.acs.org/Biomac
Conjugation of β-Sheet Peptides to Modify the Rheological Properties of Hyaluronic Acid Allison N. Elder,† Nicole M. Dangelo,† Stephanie C. Kim,† and Newell R. Washburn*,†,‡ †
Department of Chemistry, and ‡Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States ABSTRACT: Hyaluronic acid (HA) is a naturally occurring polysaccharide that is commonly used in cosmetic, wound healing, and tissue regeneration applications because of its biocompatibility and intrinsic biological activities. However, the rheological behavior of unmodified HA is not ideal for many of these. In particular, whereas chain entanglements result in an increase in viscosity, they do not prevent flow from delivery sites under zero-shear conditions. It would be of significant benefit if strategies could be developed in which robust but reversible cross-links could be incorporated within the material to allow the formation of a gel under static conditions. In developing a modification strategy, the extent of functionalization should be low to preserve the biological activities of HA. Therefore, this study focused on attaching peptides that self-assemble into β-sheets to HA to modify the viscosity at low shear rates. It was found that the peptide sequence (LS)4 forms β-sheets in aqueous media and when reacted with HA using EDC/HOBt coupling to give 6.0 ( 1.5% modification the peptide-modified HA exhibits significant increases in low-shear viscosities in comparison with the unmodified HA. However, this increase in viscosity was observed only at lower polymer concentrations and at low shear rates, suggesting that network formation is sensitive to external forces and may change at high concentrations. At higher shear rates and at higher polymer concentrations the viscosity profile of the modified HA matches that of the unmodified HA, indicating that the peptide interactions were disrupted or ineffective under these conditions. The rheology of the peptide-modified HA was also compared with samples of HA reacted with the same molar ratio of aniline, but we found that the aniline-modified HA displayed behavior comparable to that of the unmodified HA, which demonstrates that the β-sheet peptide modification technique is superior to the technique used in commercial products, such as Hyaff, at low degrees of functionalization.
’ INTRODUCTION HA is a glycosaminoglycan that is found throughout the body in the extracellular matrix but in particularly high concentrations in the vitreous humor, cartilage, and synovial fluid where it is involved in lubrication and establishing the osmotic pressure.1,2 In addition, HA is involved in the wound healing process by helping promote and moderate inflammation as well as by providing a scaffold for cell proliferation.3 Because of these properties, HA has been used for tissue regeneration studies as well as for a variety of medical and cosmetic applications.2,49 Examples of HA in the medical field include its use for injections during eye surgeries to provide lubrication and promote tissue repair as well as for knee injections for patients with osteoarthritis to help lubricate and stabilize joints.2,4 Cosmetically, HA has been used for applications such as skin lotions to promote skin restoration and help heal dryness and for skin injections for a more youthful-looking appearance.5,6,10 For HA to be used in such a diverse range of applications, it is necessary to be able to tune the rheological properties for each application. For example, a material that is suitable for topical use should gel under static conditions so that it does not spontaneously spread too thinly upon application. However, it should r 2011 American Chemical Society
also shear-thin strongly so that it can be facilely spread onto the skin. In contrast, the properties determined to be ideal for topical use would not be suitable for a material that is to be used for an application such as a skin injection or solid implantable scaffold. Therefore, it is important to develop and identify different strategies for modifying the rheological properties of HA solutions. Whereas there have been numerous strategies developed for cross-linking HA to form irreversible hydrogels,8,11,12 there have been relatively few reports of methods to tune HA rheological properties. The rheology of unmodified HA has been studied in detail. A study by Krause et al determined that the rheological properties of high-molecular-weight HA in buffer solution are comparable to those of typical polyelectrolytes in solutions of excess salt. HA displays Newtonian viscosities at low shear rates, with shear thinning occurring at higher shear rates and for more concentrated solutions. Additionally, the dilute polymer regime was determined to occur at concentrations
