Preparation and investigation of a cross-linked hyaluronan nanoparticles system
Introduction
Hyaluronic acid (HA) is a non-sulphated glycosaminoglycan that is an unbranched polysaccharide consisting of repetitive disaccharide units with reactive carboxyl groups. It is a biodegradable, biocompatible, non-toxic, non-immunogenic and non-inflammatory biomaterial; therefore, it has been used for several medical applications.
HA can be found in all tissues and body fluids of living creatures and most abundantly in the soft connective tissues. The total amount of HA in the adult human has been estimated to be 11–17 g (Laurent & Reed, 1991). The HA nanofibrous scaffold was successfully fabricated to mimic the architecture of natural extracellular matrix (Almond et al., 2006, Ji et al., 2006).
The excellent water-binding capacity of HA is responsible for retaining moisture in eyes, joints, and skin tissues (Robert, Robert, & Renard, 2010). The solution of HA is highly viscous with unique viscoelastic properties which enables its use for orthopedy (Witteveen, Sierevelt, Blankevoort, Kerkhoffs, & van Dijk, in press). Many studies have been performed to create HA as an injectable form (Salk, Chang, D’Costa, Soomekh, & Grogan, 2006), which is used to treat osteoarthritis of the knee. (Strand et al., 2006).
HA can be used as an eye-treating solution (Nakamura, Sato, Chikama, Hasegawa, & Nishida, 1997). Oral application of HA has been lately suggested, although its effectiveness needs to be demonstrated.
Several HA derivatives have been developed for drug delivery. HA has potential as a biodegradable carrier for transdermal drug delivery (Avila et al., 2008). HA has also been used as a novel depot system (Oh et al., 2010). HA in the forms of physically and chemically cross-linked hydrogels (Kim and Park, 2002, Leach and Schmidt, 2005, Li et al., 2004) has been developed as nano- and micro particulate systems (Choi et al., 2008, Segura et al., 2005) for various protein, drug (He, Zhao, Yin, Tang, & Yin, 2009), peptide (Moriyama, Ooya, & Yui, 1999) or gene (Lee et al., 2007, Luten et al., 2008) delivery.
Various methods have been developed to produce cross-linked hyaluronic acid, as hydrogels (Crescenzi et al., 2003, Masters et al., 2005), films (Liu, Shu, & Prestwich, 2005), or particulate systems (Dulong et al., 2004, Pitarresi et al., 2007). Particulate systems are usually formed in emulsion, in which the size of droplets can control the size of particles. Solvent evaporation (Lim, Forbes, Berry, Martin, & Brown, 2002), spray-drying (Esposito, Menegatti, & Cortesi, 2005) and coacervation (Vasiliu, Popa, & Rinaudo, 2005) are also well-known techniques to produce micro- or nano-sized particulate systems.
In this work, preparation of stable cross-linked HA nanoparticles is described. The particulate systems were obtained by covalently cross-linking of carboxyl groups of HA linear chain with a 2,2′(ethylenedioxy)bis (ethylamine) in the presence of water-soluble carbodiimide (CDI) in aqueous media, as described earlier (Bodnár et al., 2009). The purpose of the present study was to investigate the effect of the reaction conditions on the formation of HA nanoparticles. It was observed that the salt and HA concentrations in the reaction mixture have significant effect on the size of particles formed.
The results based on TEM, DLS and rheology experiments reveal that well-dispersed HA nanoparticles systems with spherical shape were obtained. It was found that the particle sizes and size distribution can be influenced by the concentration of HA and the salt concentration of the media.
Intra and intermolecular cross-linking processes were formed and nanosystems with broad size distribution were produced, however, the smaller particles were lost during the dialysis.
Section snippets
Materials
The HA sodium salt (MW = 4350 kDa) was obtained from Gedeon Richter Ltd., Hungary. Quality of the sodium hyaluronate met the European Pharmacopoeia (Ph. Eur.) requirements. 2,2′(Ethylenedioxy)bis(ethylamine) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (CDI) were purchased from Sigma–Aldrich, Co. The pH was adjusted with NaOH and HCl solutions as required. All other chemicals were analytical grade. Millipore-filtered water was used throughout the study.
Preparation of cross-linked hyaluronan nanoparticles
Cross-linked HA
Transmittance results
The transmittance values were measured from the reaction mixtures containing different cross-linked hyaluronan nanoparticles. These colloid dispersions were transparent or mildly opalescent systems in aqueous media. The transmittance values were between 91% and 99%.
The general trend that appears in Fig. 2a is in accordance with the Bouguer–Lambert–Beer law that transmittance values decrease due to the increasing concentrations of HA. The transmittance values were very high in wide pH range
Conclusions
In this paper, we have shown that nano-sized particles based on HA have been successfully prepared by amidation with a bifunctional amine as a cross-linking agent in the presence of carbodiimide. Transparent or mildly opalescent colloid systems were fabricated in aqueous media at room temperature.
Physico-chemical properties, including transmittance of aqueous system containing HA nanoparticles, hydrodynamic size and size distribution of hyaluronan nanoparticles were controlled by varying the
Acknowledgements
Authors thank Lajos Daróczi for TEM micrographs. The study was supported by the TAMOP 4.2.2-08/1 and GOP-2.1.1-09/A-2009-2848 and Innocsekk Plusz grants.
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