Carbohydrate Polymers 88 (2012) 799–808 Contents lists available at SciVerse ScienceDirect Carbohydrate Polymers jo u rn al hom epa ge: www.elsevier.com/locate/carbpol New hyaluroran derivative with prolonged half-life for ophthalmogical formulation Gemma Leone a,∗ , Marco Consumi b , Stefania Lamponi a , Agnese Magnani a a Department of Pharmaceutical and Applied Chemistry, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy b ALTA S.r.l.u., Via Fiorentina 151, 53100 Siena, Italy a r t i c l e i n f o Article history: Received 7 October 2011 Received in revised form 19 December 2011 Accepted 24 December 2011 Available online 24 January 2012 Keywords: Hyaluronan Phosphorylation Viscosity Enzymatic degradation a b s t r a c t Two new hyaluronan derivatives containing phosphate or cystamine groups have been synthesised to obtain tear drops with improved rheological properties and prolonged half-life. Rheological analysis, in terms of apparent viscosity, pointed out that phosphorylated hyaluronan showed promising character- istics for the foreseen application. The presence of phosphate groups along the chains, confirmed by infrared analysis, induced a strengthening in hyaluronan hydrogen bond capability thus modifying its thermal behaviour and prolonging its resistance against hyaluronidase attack. Finally, in vitro cytoxicity tests excluded any adverse effect of the developed hyaluronan derivative. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Eye drops are commonly instilled to treat a variety of ocular problems, such as dry eyes, glaucoma, infections, allergies, despite their short contact time with the eye surface, which results in reduced effects for artificial tears or low bioavailability for oph- thalmic drugs (Maurice, 1973). In fact, if the instilled fluid has a viscosity similar to that of tears, which is about 1.5 mPa s, the instilled fluids or solutes are eliminated from the tears in few min- utes (Snibson et al., 1990; Yokoi & Komuro, 2004). To increase the duration of comfort after drop instillation or to increase the bioavailability of the drugs, it is desirable to prolong the residence time for the instilled fluid. It has been shown in a number of clinical and animal studies that the retention began to increase only after the fluid viscosity exceeded a critical value of about 10 mPa s (Zaki, Fitzgerald, Hardy, & Wilson, 1986; Zhu & Chauhan, 2008). Although increasing fluid viscosity increases the residence time, it may also cause discomfort and damage to ocular epithelia due to an increase in the shear stresses during blinking. Natural macromolecules such as hyaluronan (HA), present in the vitreous body of the eye, have been proposed as viscosifying agents. Sodium hyaluronate has physical properties and a compo- sition comparable to tear glycoproteins and easily coat the corneal epithelium. The non-Newtonian behaviour of sodium hyaluronate ∗ Corresponding author. E-mail address: leone10@unisi.it (G. Leone). combines the advantage of high viscosity at rest between blinks with those of lower viscosity during blinking (Ludwig, 2005). Diluted solutions of sodium hyaluronate have been employed successfully as tear substitutes in severe dry eye disorders. The beneficial effects are attributed to the viscoelasticity, biophysical properties similar to mucins, providing a long-lasting hydration and retention. Moreover, good lubrication of the ocular surface is obtained (Ludwig, 2005). However, one of the disadvantage of actually commercialised preparations based on HA is the very short half-life due to the hyaluronidase attack, being degraded within the first 3 h. The aim of this study is the development of new hyaluronan derivatives with improved rheological properties as tear drops and with a longer half-time with respect to native hyaluronan. Two different derivatives were synthesised by the introduc- tion of phosphate and cystamine groups along the chain. Both phosphate and amino groups should increase the hydrophilicity of polysaccharide (Barbucci et al., 2005). The effect of these new functionalities on the rheological properties and the enzymatic degradation rate was evaluated as well as their cytotoxicity. 2. Materials and methods 2.1. Materials Sodium salt of hyaluronan (MW 1.2 × 10 6 ) was received as a gift from SIFI S.p.A (Italy). 0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbpol.2011.12.047