(373m) The Influence of the Organic Phase On the Synthesis of Hyaluronic Acid Nanoparticles by Nanoprecipitation

Souza, R. C., UNICAMP
Martins, F., UNICAMP


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.
(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.

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.


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.

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.


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



Mean Diameter (nm)*


Zeta Potential (mV)


239.15 ± 46.05

0.527 ± 0.106

-29.5 ± 5.5


421.98 ± 15.34

0.543 ± 0.160

-32.2 ± 7.8


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.

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.


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.


  • 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. 


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