(608b) Synthesis of Hyaluronic Acid Nanoparticles by Controlled Nanoprecipitation In Bulk Solution or Using Hydrodynamic Flow In Microfluidic Channels

1. BACKGROUND Hyaluronic acid is (HA) is a biocompatible linear polysaccharide with high molecular weight composed of disaccharide units of D-glucuronic acid and N-acetylglucosamine. Owing to its ability to retain large volumes of water, HA is used as a drug delivery system in sponges, films and nanoparticles. Nanoprecipitation has been a promising processing to obtain nanoparticles in only one step, free from oil and surfactants. In contrast with the standard nanoprecipitation procedure, in this case. HA polymer is in the aqueous phase and the organic phase is poured into the aqueous phase under stirring. The water diffuses into the organic solvent and the associated HA chains aggregate forming nanoparticles. The mechanism of nanoparticle formation is based on the water-solvent, water-polymer and solvent-polymer interactions. This experimental study presents a comparison between the HA nanoparticles produced in bulk solution and in microfluidic microchannels.

2. EXPERIMENTAL The discontinuous process was carried out according to the protocol described by Hu et. al. (2006). The carboxylic groups of glucuronic acid from HA are crosslinked with adipic hydrazide (ADH), in a reaction mediated by chloride carbodiimide (EDCl). The process occurred in a jacket glass reactor equipped with a mechanical stirrer and the temperature in the reactor was controlled at 21 ¢XC. The molar ratio between the organic solvents and HA was kept constant. HA with molecular weight 2,000 Da was used in its salt form HNa from a 1% solution. The experiments in continuous process were carried out in a microchannel system. Typically, aqueous phase with HA, ADH and EDCl was pumped into the main channel. At the same time, the organic phase was fed into the inlets of the two branch channels using two identical but separate syringes by another pump. The processes were performed under flows that provided the same volume ratio of the discontinuous processes. The processes were performed with acetone, isopropyl alcohol (IPA) and ethanol as the organic phases. The results show the performance of both systems to the synthesis of HA nanoparticles, in terms of HA nanoparticles size, polidyspersity and zeta potential.

3. RESULTS AND DISCUSSION In both cases, the size of particles was in the range of 200-400 nm. In the discontinuous process, HA molecules are in the aqueous phase and the organic phase is poured into it under stirring. The water diffuses into the organic solvent and the associated HA chains aggregate forming nanoparticles. On the other hand, in the continuous process, nanoprecipitation occurred in laminar flow inside a microchannel, formed at the interface between the organic phase (polymer non-solvent) and water, in which HA was solubilized. In the presence of IPA or ethanol smaller nanoparticles were formed in the continuous process, while in the discontinuous process the smallest nanoparticles were obtained with acetone. Stable nanoparticles were obtained in both types of process and for all the organic solvents used, what is confirmed by the low zeta potentials. In continuous process, the mean diameter correlates with the inverse of the affinity between water and organic phase. The higher the affinity between the organic and the aqueous phases, the lower the interaction and Hansen solubility parameters are. Therefore, the ability of the non-solvents to HA dehydration decreases from ethanol, IPA to acetone, while the mean diameter increases from ethanol, IPA to acetone. On the other hand, in the discontinuous process, the mean diameter correlates with the inverse of surface tension. IPA presents the lower surface tension. Therefore, water migrates faster to IPA compared to acetone and ethanol, and because of that faster and uncontrolled water displacement, larger particles were prepared with IPA compared to ethanol and acetone.

4. CONCLUSIONS The results obtained show that the production of HA by nanoprecipitation and crosslinking with ADH is promising, due to the simplicity of the process by not requiring the later stages of downsizing and being capable of been conducted in large-scale. The mean diameter and polydispersity of HA nanoparticles obtained in continuous process are controlled by the solubility of water in the organic solvent. On the other hand, the surface tension controls the mean diameter and polydispersity in the discontinuous process. In both cases, the crosslinked particles were stable under refrigeration at a minimal of two months. These results are important for applications involving controlled release of drugs, scaffolds and tissue regeneration.

5. REFERENCES 1. PRESTWICH, G.D.; MARECAK, D.M.; MARECK, J.F.; VERCRUYSEE, K.P.; ZIEBELL, M.R. (1998), "Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives". Journal of Controlled Release, 53, pp. 93-103. 2. HU, Z.; XIA, X.; TANG, L. (2006), "Process for synthesizing oil and surfactant-free hyaluronic acid nanoparticles and microparticles". US Patent 20060040892 A1. 3. ZHANG, S.; YUN, J.; SHEN, S.; CHEN, Z.; YAO, K.; CHEN, J.; CHEN, B.; (2008) "Formation of solid lipid nanoparticles in a microchannel system with a cross-shaped junction". Chemical Engineering Science, 63, pp. 5600-5605.