Characterization of Methylprednisolone Esters of Hyaluronan in Aqueous Solution: Conformation and Aggregation Behavior Anna Taglienti, ² Massimiliano Valentini, ‡,§ Paolo Sequi, ‡ and Vittorio Crescenzi* ,² Department of Chemistry, University “La Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy, Experimental Institute for Plant Nutrition, Via della Navicella 2-4, 00184 Rome, Italy, and Institute for Biomedical Engineering and Department of Materials, Swiss Federal Institute of Technology and University of Zurich, Moussonstrasse 18, CH-8044 Zurich, Switzerland Received December 9, 2004; Revised Manuscript Received February 9, 2005 Methylprednisolone steroid esters of hyaluronan differing in degree of functionalization and molecular weight were investigated in aqueous solution. Conformation and aggregation phenomena were elucidated by means of circular dichroism, viscometry, rheology, and nuclear magnetic resonance, mainly by 1 H pulsed field gradient (PFG) NMR, which allows the determination of the diffusion coefficient of the species under investigation. The functionalization of hyaluronan with the steroid induces a reduction of the molecular volume, as a consequence of intramolecular hydrophobic interactions. For concentrated samples we have observed the coexistence of unimolecular collapsed chains and of aggregates, the latter disappearing upon dilution. The methylprednisolone ester of lower molecular weight hyaluronan has a larger molecular volume than its higher molecular weight analogue, even though still smaller than the underivatized polymer. This effect can be explained with the reduced flexibility of the polymer backbone probably impairing intramolecular interactions. Introduction Hyaluronan (HA) is a glycosaminoglycan 1,2 with important biological functions. 3 It is one of the major components of the extracellular matrix and participates in several relevant biological processes, such as cell motility, 4 cell differentia- tion, 5 wound healing, 6 and cancer metastasis. 7 As a conse- quence of its high degree of biocompatibility, biodegrad- ability, nonimmunogenicity, and viscoelastic properties, HA is currently used in viscosurgery, 8 ophthalmic surgery, 9 and osteoarthritis therapy. 10,11 The biological relevance and the versatility of HA structure make this molecule an attractive building block for the preparation of polymers with potential biomedical applica- tions. 12 Many chemical modifications of the native HA structure 13 have been obtained; for example, partial or total esterification of the carboxylic groups produces biomaterials with interesting rheological properties and with high pro- cessability for preparing films and microspheres. 14 The covalent binding of drugs 15 to the carboxylic functions has received considerable attention since this procedure gives a polymeric prodrug as product. One of the most interesting and promising polymeric prodrugs derived from HA is obtained from the proper combination of hyaluronan with the steroid methylprednisolone. 16 The latter was covalently bound to HA by esterification of the hydroxyl groups in C-21 position of the steroid with the carboxyl groups of the glucuronic acid moieties of hyaluronan. 17 Prior to any in vivo investigation of the hyaluronan/ methylprednisolone system, a structural characterization in solution of such derivative was necessary since it is known that physical and chemical properties can often influence drastically the efficiency of polymeric drug carriers in terms of pharmacodynamics, pharmacokinetics, drug delivery, and biodegradability. In this paper we report a structural study of the hyaluronan/ methylprednisolone derivative for which the influence of some key parameters such as degree of functionalization, polymer concentration, and molecular weight has been investigated and elucidated. Experimental Section Materials. D 2 O was purchased from Acros Organics and was used without further purification. Sodium hyaluronate of high (about 600 000, fraction Hyalectin) and medium (about 200 000, fraction Hyalastine) molecular weight, and hyaluronan methylprednisolone esters of high and medium molecular weight, HYC141 and HYC41, respectively, were supplied by Fidia Farmaceutici S.p.A. (Abano Terme, PD, Italy) and used without further purification (Figure 1). HYC141 derivatives have 60%, 45%, or 16% of the carboxylic groups of glucuronic acid functionalized with the steroid, denoted HYC141p60, HYC141p45, and HYC141p16, respectively, while the HYC41 derivative has 45% of carboxyl functions esterified, denoted HYC41p45. The remaining carboxylic groups are in the sodium salt form. It should be noted that quite naturally the charge density decreases as the degree of functionalization increases. * To whom correspondence should be addressed: tel +39 6 49913630; e-mail vittorio.crescenzi@uniroma1.it. ² University “La Sapienza”. ‡ Experimental Institute for Plant Nutrition. § Swiss Federal Institute of Technology and University of Zurich. 1648 Biomacromolecules 2005, 6, 1648-1653 10.1021/bm049221d CCC: $30.25 © 2005 American Chemical Society Published on Web 03/17/2005