The Influence of the Organic Phase On the Synthesis of Hyaluronic Acid Nanoparticles by Nanoprecipitation

1. BACKGROUND

The hyaluronic acid (HA) is a natural mucopolisaccharide composed of disaccharide units of D-glucuronic acid and N-acetylglucosamine. In humans, HA is the major component of the vitreous humor in the eyes, the synovial fluid in joints and the umbilical cord. Due to its physical, chemical and biological properties, HA has a wide range of pharmaceutical, medical and cosmetics applications (Holmström and Ricici, 1967 apud Kogan et. al., 2007).

The typical chemical modifications of HA involve carboxylic groups and/or hydroxyl groups of the main chain by linking adjacent polymer chains through crosslinking agents (Prestwich, 2010).

Yun et. al. (2004) studied the synthesis of sodium hyaluronate (HNa) particles produced by W/O emulsification and crosslinked with ADH and chloride carbodiimide (EDCI) as a catalyst in aqueous media. The experimental results showed that the emulsifying technique produces polydisperse particles with sizes ranging from nanometers to micrometers. In addition, the emulsification process requires the use of surfactant and the subsequent withdrawal of the oil by washes with isopropyl alcohol, which causes significant losses of particles in the process.

Aiming to overcome these difficulties, Zhibing et. al. (2006) patented a process for the production of HA nanoparticles free from oil and surfactants. The process includes the replacement of the oil phase by the organic solvent acetone as well as the nanoprecipitation of sodium hyaluronate (HNa) and the crosslinking with ADH and EDCI. The produced HA particles were insoluble in water and they had uniform sizes around 200 nm.

The mechanism of nanoparticle formation depends on the water-solvent, water-polymer and solvent-polymer interactions. A relationship between the physicochemical properties of the aqueous and organic phases could explain the formation, size and polydispersity of nanoparticles produced by nanoprecipitation (Garlindo-Rodriguez et. al., 2004).

Within this context, this work aims to study the influence of the nature of the organic solvent on the mean diameter, polydispersity and zeta potential of the HNa particles produced by nanoprecipitation and crosslinked with ADH and EDCI. Experimentally the study was carried out using polar protic solvents such as ethanol and  isopropyl alcohol (IPA) as well as the polar aprotic solvent acetone. The importance of the second addition of the solvents during the process was also investigated.

2. EXPERIMENTAL

The production of nanoparticles was carried out in a jacket glass reactor of 400 mL, equipped with a mechanical stirrer (model TE-039/1, Tecnal). The temperature in the reactor was controlled by circulating water from a thermostat bath (model TE-184, Tecnal). The molar ratio between the organic solvents and HA was kept constant. As the MW and density of the used solvents are similar, there were not significant differences in HA concentration in the experiments.

The nanoparticles were obtained by nanoprecipitation followed by chemical crosslinking of HNa with ADH and EDCI, according to the protocol described by Zhibing et. al. (2006). Initially, 8 grams of HNa (1%) were added to 80 mL of Milli-Q water. Then, it was added, gently, 137 mL of the organic solvent. The system was kept under agitation at 300 rpm and 21°C for 2 h. ADH (0,04g) and EDCI (0,08g) in 2 mL of Milli-Q water were added and the system was kept under the same agitation at 21°C during 24 h. A second volume of organic solvent (132 mL) was added to the medium, continuing the reaction for further 20 h. Then, the produced particles were separated through retention in a 10.000 kDa pore ultrafiltration membrane. Both the filtrate and the retained particles were analyzed. The mean diameter, polydispersity and zeta potential were measured using a Zetasizer Nano Series ZS (Malvern Instruments), with fixed angle of 173°. All the experiments were performed in duplicate. Size distribution by number was considered in the analysis.

3. RESULTS AND DISCUSSION

In contrast to the standard nanoprecipitation procedure for hydrophobic polymers, HA molecules are in the aqueous phase and the organic phase is the polymer non-solvent. The water diffuses into the organic phase and the HA chains precipitate forming particles. The mechanism of nanoparticle formation is based on the water-non-solvent, water-polymer and non-solvent-polymer interactions. HA is completely soluble in water and insoluble in the organic solvents. Therefore, the water-non-solvent-HA interactions at the interface were the only analyzed effects aiming to explain the mean diameter, polydispersity and zeta potential of the HA particles.

The experimental results (Table 1) showed that HA nanoparticles were obtained with the three studied solvents, but their sizes varied from 100 to 400 nm depending on the solvent. Therefore, the organic solvent plays an important role in the rate of particle formation and polydispersity.

Table 1 – Mean diameter, polydispersity and zeta potential of HA particles obtained with the different non-solvents: ethanol, IPA and acetone

 

Solvents	Mean Diameter (nm)*	Polydispersity	Zeta Potential (mV)
Ethanol	239.15 ± 46.05	0.527 ± 0.106	-29.5 ± 5.5
IPA	421.98 ± 15.34	0.543 ± 0.160	-32.2 ± 7.8
Acetone	120.44 ± 12.14	0.271 ± 0.106	-22.6 ± 4.0

                        * Mean diamenter determined by Z-average.

 

As can be seen in Table 1, colloidal stability indicated by the zeta potential in the range of -22 to -32 was obtained for HA nanoparticles prepared with all the studied non-solvents. These particles were stable under refrigeration for at least two months. However, smaller nanoparticles (120.44 nm) and less polydispersed ones (0.271) were obtained with acetone. Such differences could be analyzed through the degree of affinity between water and the organic solvents, the conformation of HA and the surface tension at the water-organic phase interface.

Galindo-Rodriguez et. al. (2004) presented the Hansen solubility and interaction parameters for some solvents used in nanoprecipitation. The higher the affinity between the organic and the aqueous phases, the lower the interaction (C) and Hansen solubility (d) parameters are. It could be observed that the Hansen solubility and interaction parameters increase in the following order: ethanol < IPA < acetone. Therefore, the ability of the non-solvents to HA dehydration decreases from ethanol, IPA to acetone.

The surface tensions at the water-organic phase interface vary depending on the organic solvent used. They increase from IPA, ethanol to acetone. These differences influence the displacement of water to the organic phase and the precipitation rate of HA. 

The results show that the mean diameters and polydispersity do not correlate with the affinity, but they correlate with the inverse of surface tension. The lower the surface tension is, the larger and more polydispersed the particles are. The lowest surface tension at the interface IPA-water leads to a high rate of HA dehydration and precipitation. The faster formation leads to an alleatory growth of the precipitated cores, generating nanoparticles with larger sizes and polydispersity. The most controlled water displacement to acetone generates HA nanoparticles with lower mean diameter and polydispersity. These results infer that mass transfer of water between the solvents controls the mean diameter and polydispersity of HA nanoparticles.

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 surface tension, which determines the rate of water displacement to the organic phase, controls the mean diameter and the polydispersity of HA nanoparticles in the discontinuous process. In all 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

• Galindo-Rodriguez, S.; Allemann, E.; Fessi, H.; Doelker, E.; Pharmaceutical Research, v.21, n.8, p.1428-1439, 2004.
• KOGAN, G.; SOLTÉS, L.; STERN, R.; Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnol. Lett., v. 29, p. 17-25, 2007.
• Prestwich, G. D.; Biomaterials from chemically-modified hyaluronan. Available in: <http://www.glycoforum.gr.jp/science/hyaluronan/HA18/HA18E.html>. Accessed on 22/03/2010.
• Yun, Y. H.; Goetz, D. J.; Yellen, P.; Chen, W.; Hyaluronan microspheres for sustained gene delivery and site-specific targeting, Biomaterials, 25, p.147-157, 2004.
• Zhibing; Hu; Xiaohu; Xia; Liping; Tang; Process for synthesizing oil and surfactant-free  hyaluronic acid nanoparticles and microparticles. US Patent 20060040892 A1.