Eco-friendly microwave-assisted protocol to prepare hyaluronan-fatty acid conjugates and to induce their self-assembly process

Article abstract of DOI:10.1016/j.carbpol.2016.02.001

An environmentally sustainable and energy-efficient synthetic process has been developed to prepare hyaluronan-based nano-sized material. It consists in a microwave-promoted acylation of the hydroxyl function of the polysaccharide with natural fatty acids, performed under solvent-free conditions. The efficient interaction of the solid reagents with the MW radiation accounts for the obtained high yielded products. The self-assembly process of the obtained compounds very fast occurred in an aqueous medium under MW-radiation, thus allowing the development of a green protocol for the nano-particles preparation.

Full text of DOI:10.1016/j.carbpol.2016.02.001

Carbohydrate Polymers 143 (2016) 84–89
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Eco-friendly microwave-assisted protocol to prepare hyaluronan-fatty
acid conjugates and to induce their self-assembly process
Enrica Calcea, Flavia Anna Mercurioa, Marilisa Leonea, Michele Savianob,
Stefania De Lucaa,∗
a Institute of Biostructures and Bioimaging, National Research Council, 80138 Naples, Italy
b Institute of Crystallography, National Research Council, 70126 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
An environmentally sustainable and energy-efficient synthetic process has been developed to prepare
hyaluronan-based nano-sized material. It consists in a microwave-promoted acylation of the hydroxyl
function of the polysaccharide with natural fatty acids, performed under solvent-free conditions. The
efficient interaction of the solid reagents with the MW radiation accounts for the obtained high yielded
products. The self-assembly process of the obtained compounds very fast occurred in an aqueous medium
under MW-radiation, thus allowing the development of a green protocol for the nano-particles prepara-
Received 1 December 2015
Received in revised form 26 January 2016
Accepted 1 February 2016
Available online 3 February 2016
Keywords:
Hyaluronan-based material
Renewable sources
tion.
© 2016 Elsevier Ltd. All rights reserved.
Microwave activation
Solvent-free synthesis
Nanoparticle preparation
Biocompatible process
1. Introduction
a quite small size (Calce et al., 2015). The smallest aggregates have
been considered for a potential employment as stable and effective
HA, member of the glycosaminoglycan family, consists of a
linear polysaccharide with repeating beta-1,4-linked d-glucuronic
acid (GlcA) and beta-1,3-linked N-acetyl-d-glucosamine (Glc-
NAc) disaccharide units. Due to its inherent biocompatibility and
biodegradability, as well as its susceptibility to chemical modifi-
cation, several HA-based biomaterials with promising biomedical
potential have been developed (Lee, Mok, Lee, Oh, & Park, 2007;
Manju & Sreenivasan, 2011; Mironov et al., 2005; Peer & Margalit,
2004). In particular, by chemical conjugation of hydrophobic moi-
eties to the hydrophilic HA backbone, several nano-aggregates
were prepared and studied as nano-sized drug delivery system for
cancer treatment (Cho et al., 2011, 2012; Choi et al., 2009, 2010;
Ganesh, Iyer, Morrisey, & Amiji, 2013; Huang et al., 2014; Schanté,
Zuber, Herlin, & Vandamme, 2011).
drug delivery system for biomedical applications. The employed
synthetic procedure uses solvent-free conditions and microwaves
of domestic oven for activation, in other words it consists in a MW-
induced solid-state reaction that provides the great advantage of
a clean, fast and energy-efficient synthetic method. Compared to
the solution-phase MW synthesis, the solid-state MW-activation
has experienced a narrow application into the field of organic
chemistry, this is due to the less comprehensive MW-solid inter-
actions (Kappe, 2013; Kitchen et al., 2014). Moreover, there are
serious difficulties associated with monitoring temperature condi-
tions inside the solid. On the other hand, it has been hypothesized
an increased mobility of polar solid substrates under MW radiations
that increases the heating rate. Therefore, milder reaction condi-
tions can be used for chemical process to produce high yielded final
products (de la Hoz, Diaz-Ortiz, & Moreno, 2005).
Herein, we report on a microwave-assisted synthetic protocol to
prepare HA-fatty acid conjugates characterized by defined degrees
of substitution. The reaction parameters were deeply investigated
and a tuned synthetic protocol was developed to prepare HA-oleate
and HA-linoleate. This procedure not only shortens the reaction
time, but also improves its yield and reproducibility. Moreover,
also the self-assembly process of the polysaccharides conjugates
In this regard, we have recently developed amphiphilic
HA-derivatives by chemical conjugation of natural fatty acids to
the backbone of the polysaccharide, and the obtained materials
were able to self-assemble, forming spherical nano-particles with
∗
Corresponding author.
E-mail address: stefania.deluca@cnr.it (S. De Luca).
http://dx.doi.org/10.1016/j.carbpol.2016.02.001
0144-8617/© 2016 Elsevier Ltd. All rights reserved.

E. Calce et al. / Carbohydrate Polymers 143 (2016) 84–89
85
was driven by the MW irradiation. The obtained materials were
characterized for their properties, like the degree of substitution,
the particle size, zeta potential and also for their morphology.
for these compounds have been previously reported (Calce et al.,
2015).
2.1.3. FT-IR characterization
All modified hyaluronan samples were analyzed by FT-IR spec-
troscopy. The FT-IR spectra were recorded on a Jasco FT/IR 4100
spectrometer. Samples were ground into a fine powder using an
agate mortar before being compressed into KBr discs. The char-
acteristic peaks of IR transmission spectra were recorded at a
2. Experimental
2.1. Synthesis and characterization of HA-fatty acids conjugated
2.1.1. Synthesis
resolution of 4 cm−1over a wavenumber region of 400–4000 cm−1
The bands relevant for the structural organization are: HA-oleate
(1d): FT-IR (cm−1): 3428 ꢀ(O H), 2923 ꢀ(C H), 1737 ꢀ(C
fatty acid ester), 1648 ꢀas(COO−), 1413 ꢀas(COO−), 1078 ꢀ and
1040 ꢀ(COC)glycosidic
ring. HA-linoleate (2d): FT-IR (cm−1):
.
Fatty acid anhydrides were synthesized as follows. The appro-
priate fatty acid (10 mmol) was dissolved in dichloromethane
(2 mL), the solution was cooled in an ice-water bath and stirred
vigorously under argon atmosphere. The dicyclohexylcarbodi-
imide (5 mmol), previously dissolved in the minimum volume of
dichloromethane, was added and the stirring was continued at ice
bath temperature for 2 h. The white solid N,Nꢀ-dicyclohexylurea
was removed by filtration and the solvent was evaporated in vac-
uum to give the final anhydride.
O
bond
3419 ꢀ(O H), 2926 ꢀ(C H), 1734 ꢀ(C O fatty acid ester), 1648
ꢀas(COO−), 1415 ꢀas(COO−), 1078 ꢀ and 1040 ꢀ(COC)glycosidic bond
.
ring
By using an agate mortar, hyaluronan and the appropriate fatty
acid anhydride, at the same weight ratio, were manually milled in
presence of catalytic amount of K2CO3 to obtain several hyaluronan
derivatives.
2.2. Preparation and characterization of HA-fatty acids
nano-particles
2.2.1. Synthesis of HA-fatty acids nano-particles
The samples, placed in a 0.5–2 mL microwave vial, were irradi-
ated for 2 min in a microwave oven (Initiator, Biotage Sweden AB,
Uppsala, Sweden) under the following conditions:
HA-oleate and HA-linoleate nano-particles were prepared as
follow: 1 mg of the respective conjugate was dissolved in 1 mL of
H2O 0.9% wt NaCl in a 0.5–2 mL microwave vial. Then, the solution
was irradiated by microwaves at a constant power of 40 W for 10 s
and stirred at 900 rpm.
- 80 ◦C, P max = 200 W (compounds 1a and 2a);
- 120 ◦C, P max = 220 W (compounds 1b and 2b);
- 160 ◦C, P max = 250 W (compounds 1c and 2c),
- 200 ◦C, P max = 300 W (compounds 1d and 2d);
- 100 W, T max = 53 ◦C (compounds 1e and 2e);
- 200 W, T max = 81 ◦C (compounds 1f and 2f).
All solutions were filtered through a membrane filter (pore size
0.40 ␮m, Millipore).
2.2.2. Size distribution and zeta potential of nano-particles
The particle size distribution and zeta potential of the HA-fatty
acid nano-particles were measured at 25 ◦C by dynamic light scat-
tering (DLS) technique with a Malvern Zetasizer (Nano ZS, Malvern
Instruments, Westborough, MA) with NIBS optics.
After cooling at room temperature, the obtained solid was
dissolved in water, placed in a 250 mL separatory funnel and
extracted with ethyl acetate in order to remove the unreacted fatty
acids. Subsequently, the aqueous layer was neutralized by adding
0.5 N HCl solution in water and then dialyzed (membrane cut off
6000–8000 Da) for 1 day in Milli-Q water.
The scattered light was measured at an angle of 173◦ and was
collected on an autocorrelator. The hydrodynamic diameters (d) of
micelles were calculated by using the Stokes–Einstein equation.
All data were averaged over three measurements.
The stability of HA-fatty acid conjugates was evaluated by size
change over the course of 7 days at room temperature.
The final product was collected after lyophilization process.
2.1.2. NMR characterization
1D [1H] NMR spectra were recorded for HA-oleate and HA-
linoleate in the temperature range 298–303 K either on a Varian
Unity Inova 400 MHz NMR spectrometer equipped with z-axis
pulsed-field gradients and a triple resonance probe or a Var-
ian 600 MHz spectrometer with a cold probe. To prepare NMR
samples, compounds were dissolved in 600 ␮L of D2O (99.9% D,
Sigma–Aldrich, Milan, Italy). 1D [1H] spectra were acquired with
a relaxation delay of 2 s and 512–2048 scans, without water sup-
pression. Spectra were processed with Varian software (VNMRJ).
The water signal was set at 4.75 ppm for proton chemical shifts
referencing.
2.2.3. Transmission electron microscopy
Transmission electron microscopy observation was performed
on a microscope Tecnai G2 Spirit TWIN operating at an acceleration
voltage of 120 kV. The specimen was prepared as follows. One drop
of dilute latex was delivered on a copper EM grid covered with a
thin holey carbon film and dried at room temperature.
3. Results and discussion
In a previous study we developed a MW-assisted esterifica-
tion reaction to prepare hyaluronan derivatives. It consisted in a
solvent-free process for generating conjugates of the polysaccha-
ride, via OH esterification with natural fatty acids (Calce et al.,
2015). We also tried to modulate the degree of hyaluronan hydroxyl
functionalization by varying several reaction conditions, like the
MW time exposure and the reacting amount of fatty acid anhydride
and polysaccharide.
Unfortunately, no control on the synthetic process was regis-
tered by acting on these parameters.
However, moving from a domestic MW oven to a MW source
apparatus, specifically designed for laboratory use, we decided to
investigate the effects of the reaction temperature on the pro-
cess efficiency. In particular, we studied the synthetic process to
The degree of substitution (DS) was estimated from analysis of
1D [1H] experiments by calculating the ratio (Ia/3)/(Ib/2) where Ia
indicates the integral of the peak corresponding to the CH3 protons
of the newly introduced fatty acid chains (close to 0.86 ppm) and Ib
the area (=integral) covering the peaks of sugar anomeric protons
in the spectral region between 4.37 and 4.65 ppm. Each integral
was normalized for the corresponding number of protons in one
HA disaccharide unit.
For HA-linoleate (2d) and HA-oleate (1d) 2D [1H, 1H]
TOCSY (Griesinger, Otting, Wuthrich, & Ernst, 1988) experiments
(2048*256 total data points and 64 scans pet t1 increment, 70 ms
mixing time) were acquired as well. Proton chemical shifts values

86
E. Calce et al. / Carbohydrate Polymers 143 (2016) 84–89
Scheme 1. Schematic view of the preparation of HA-conjugates.
prepare HA-oleate and HA-linoleate, since their aggregates
revealed a particularly interesting narrow size (Calce et al., 2015).
We designed several experiments characterized by a controlled
temperature and unconstrained microwave irradiation power, and
the temperature values were selected taking in consideration the
oil bath temperature, generally used for conventional heating (oil
bath T = 160 ◦C for 15 min). Moreover, being our reaction performed
in absence of solvent, we had to explore a range of temperature,
since we could not refer to the solvent settings generally transferred
from the traditional heating to a MW-activated process (Kappe,
2004).
Oleic and linoleic acids activated as anhydrides were mechan-
ically milled with the hyaluronan in the presence of catalytic
amount of K2CO3 (Scheme 1). The obtained mixture was placed
into a MW reactor and a selected temperature value was applied
for 2 min (Table 1). In order to be as reliable and reproducible as pos-
sible, the same experimental conditions (reactants amount, vessel,
etc.) were always applied. After cooling, each sample was treated
as already reported and fully characterized by FT-IR and NMR spec-
troscopy.
Typical spectra of HA-oleate and HA-linoleate (a spectrum of
the native HA is also included for comparison), are shown in Fig. 1
and clearly revealed, in the region featuring the carboxylic groups
(1740–1600 cm−1), the occurred esterification of the employed
fatty acid with the appearance of a new band in the C O ester
stretching region (1735–1740 cm−1). By evaluating the intensity of
this band, the samples resulted to be characterized by an increased
degree of substitution, if compared to FT-IR spectra of the same
conjugates prepared with conventional oil bath heating and also
with domestic MW oven irradiation (Calce et al., 2015). In partic-
ular, these data were confirmed by NMR analysis performed on
HA-oleate and HA-linoleate samples.
acid chains, that in spite of signal loss due to aggregation, is always
clear in the NMR spectra and the peak from anomeric HA protons
(Fig. 3). In a few compounds (Table 1), the anomeric proton sig-
nals in the NMR spectra resulted too close to the water peak to
allow a reliable evaluation of the integrals, thus the DS could not
be precisely estimated.
To better understand the obtained results, it is worth remem-
bering that MW activation uses the ability of some compounds
(liquids or solids) to transform electromagnetic energy into heat.
In major details, the energy transmission is produced by dielectric
losses, which is in contrast to conduction and convection processes
observed for the conventional heating. Hence MW heating is volu-
metric and fast, being the whole reaction mixture simultaneously
heated. On the basis of these considerations, we expected that our
solid state mixture is more efficiently activated by MW-radiation
compared to the conventional heating, which slower occurs from
the surface to the inner.
Moreover, MW-activation avoids the degradation of the mate-
rial in contact with the vessel wall. All these arguments supported
the increased yield observed for all the prepared hyaluronan con-
jugates, in terms of the increased efficiency of the polysaccharide
functionalization. In this regard, it is worth noting that the domestic
1a
1647,88
1736,58
As we have already reported (Calce et al., 2015), after insertion
of an acyl chain, NMR spectra of the HA-adducts become principally
affected by line broadening rather than chemical shifts changes. In
detail a few chemical shifts variations can be obviously seen and
involve mainly signals from HA, whereas excessive enlargement of
lines provokes low signal intensity for the hydrophobic acyl chains
from oleate and linoleate moieties (Fig. 2). In fact, due to aggrega-
tion, only a few of the expected signals from oleate and linoleate
can be clearly identified in the 1D [1H] spectra (Figs. 2 and 3). A
degree of substitution for most of the synthesized molecules could
be evaluated by NMR spectroscopy (Calce et al., 2015) by integrat-
ing the peak corresponding to the CH3 methyl group of the fatty
HA
1640,71
4000
3000
2000
1000
Wavenumber (cm-1)
2a
Table 1
Reaction conditions and DS values of the obtained HA-conjugates.
1647,88
1733,69
Entry
Reaction conditions
Degree of substitution (%)
HA-oleate (1)
HA-linoleate (2)
HA
a
b
c
d
e
f
80 ◦
120 ◦
160 ◦
200 ◦
100 W
200 W
C
C
C
C
3.40
4.00
3.00
5.3
n.d.
n.d.
5.3
1640,71
3.40
3.90
5.9
4.69
3.76
4000
3000
2000
1000
2 min
Wavenumber (cm-1)
Fig. 1. Transmittance FT-IR spectra of HA conjugates.

E. Calce et al. / Carbohydrate Polymers 143 (2016) 84–89
87
All the performed experiments at different temperatures
showed a very similar final yield, estimated in terms of DS (Table 1).
Since they were characterized by high initial power, we decided to
investigate the effect of this parameter, to tune the experimental
conditions of the process under investigation. In general, MW-
induced solid state reactions are far less comprehensive than those
in solution, due to the complexity of heating process and also to
the significant difficulties associated with monitoring the temper-
ature inside the sample (Kappe, 2013; Tang et al., 2014). Starting
from these considerations, we performed several experiment by
changing the MW irradiation power, while living unconstrained
the temperature (Fig. 4). We found that a quite low power was
necessary for obtaining polysaccharide conjugates with a DS value
already observed for the reaction performed at high temperatures
(Table 1).
HA signals
Fatty acid
signals
A
HA acetyl
group
B
These results confirmed that the hyaluronan esterification reac-
tion is very efficient, but does not increase the degree of substitution
by varying the reaction parameters.
The temperature values registered by the MW-radiation system
are quite low, if compared to those necessary by the conventional
heating (oil bath T = 160 ◦C for 15 min), it suggested that heating
induced by MW is very efficient (Fig. 4) (Rodriguez et al., 2015).
In other words, the interaction between the material and the
MW radiation very efficiently induce the reactants transforma-
tion and for these same reasons it can involve a selective heating
of same reagents compared to others. In particular, our reaction
mixture consists of two polar/ionic components, like the hyaluro-
nan and the potassium carbonate that very efficiently absorbs the
MW radiations, and the less polar fatty acid anhydride which is
expected to be less sensitive, on the basis of the MW activation
principles. Therefore, we can hypothesize a hot spot solvent-free
4.6
3.8
3.0
2.2
1.4
0.6
1H ppm
Fig. 2. 1D [1H] NMR spectrum of HA-linoleate (2d) (A) and HA-oleate (1d) (B).
MW oven is characterized by a low repeatability of the experimen-
tal conditions, due to the essentially random electric field pattern
applied, and it is very difficult to accurately take under control the
power delivered into the reaction vessel. It can explain the bet-
ter results reached by employing MW apparatus (Biotage Initiator),
specifically designed for chemical reactions.
Moreover, for reactions performed in polar solvent, the MW
radiations are strongly absorbed by the solvent, so that the thermal
energy is still transmitted to reagents by conventional mechanism.
In case of solid state reactions, since a solvent that could transfer
energy/heat lacks from the reaction mixture, the radiation can only
be absorbed by the reactants.
Therefore, our process is effectively MW-assisted, in such mech-
anism different from what happened with the oil bath, where the
heating is transferred to reagents from the vessel wall (de la Hoz
et al., 2005).
90
1e
2e
80
70
60
50
40
30
MW irradiation power 100 W
20
10
0
0
1
2
3
Time (min)
90
80
70
60
50
40
30
20
10
0
1f
2f
HA
Anomeric
protons
MW irradiation power 200 W
Fatty-acid
CH3
0
1
2
3
4
5
1H ppm
93.44
6.56
Time (min)
Fig. 3. 1D [1H] NMR spectrum of HA-linoleate (2e), measured integrals are indicated
on the corresponding peaks.
Fig. 4. Profile of the time vs the temperature at a fixed power value.

88
E. Calce et al. / Carbohydrate Polymers 143 (2016) 84–89
1,0
HA-Oleate
HA-oleate
HA-oleate
15
10
5
0,8
0,6
0,4
0,2
0,0
0
0,1
10
1000
Time (μs)
100000
1E7
100
1000
Size (d. nm)
10000
HA-linoleate
15
10
5
1,0
HA-Linoleate
HA-linoleate
0,8
0,6
0,4
0,2
0,0
0
0,1
10
1000
Time (μs)
100000
1E7
100
1000
Size (d. nm)
10000
Fig. 5. Size distribution vs intensity and intensity correlation of HA-oleate nano-particles (upper panel) and of HA-linoleate nano-particles (lower panel).
process, which reduces the decomposition of compounds, like the
fatty acid anhydrides, thermally unstable.
used to prepare the nano-particles of HA-oleate and HA-linoleate,
based on the emulsion evaporation of organic/water system sol-
vent, was replaced by a MW-assisted aqueous route. It allowed us
to replace the chloroform with a green solvent like the water, which
also greatly adsorbs the MW radiation and transfers the thermal
energy to other compounds (Yang et al., 2015). The obtained nano-
particles resulted stable under physiological conditions (7 days),
thus proving that the developed method was plenty successful. At
the best of our knowledge, this is the first example of protocol
which employs MW radiation to promote the spontaneous self-
association of amphiphilic polymers. All other methods employed
for a controlled MW-assisted nanoparticle synthesis are, however,
at an initial stage, in terms of understanding of the process mech-
anism and optimization of its parameters (Yang et al., 2015; Duan,
Wang, & Li, 2015).
The nano-particles of HA-oleate and HA-linoleate were also pre-
pared by employing a MW irradiation, the samples were dissolved
in an aqueous solution of NaCl 0.9% and irradiated with a power
of 40 W for 10 s. The obtained aggregates revealed a mean diame-
ter of 80.57 nm for HA-oleate and of 114.1 nm for HA-linoleate and
a narrow size distribution (Fig. 5), as confirmed by the measured
polydispersity values (Table 2).
Among the different methods developed for preparing nano-
particles of natural polysaccharide, the aqueous self-assembly
process is one of the most successful for amphiphilic polymer.
It allows the spontaneous association between hydrophobic moi-
eties, hence a core–shell structural organization leads to the
formation of micellar aggregates (Yang, Han, Zheng, Dong, & Liu,
2015). Generally, the ultrasonic bath is used to speed up this pro-
cess, by breaking molecular interactions to allow the formation of
new interactions, that can minimize the interfacial free energy of
the system (Yang et al., 2015).
4. Conclusions
We tuned a MW-assisted reaction in order to prepare hyaluro-
nan conjugates by acylation of the polysaccharide alcoholic
functions with oleic and linoleic acids, activated as anhydride.
The material-radiation interaction is believed responsible of the
efficient thermal effect. Indeed, this effect is not masked or limited
by the solvent, since the reaction is performed under solvent-free
conditions. Moreover, harsh reaction parameters could be avoided
under MW irradiation, in fact we demonstrated that with a power
of 100 W for 2 min (maximum temperature reached 50 ◦C) a good
degree of hyaluronan functionalization already occurred. When
compared to modified hyaluronan obtained under conventional
In the same way, we have hypothesized that the orientation
induced on polar molecules by the microwave radiations could
favor the micellar self-aggregation, so that the previous method
Table 2
Characteristic parameters of HA-fatty acid nano-particles.
HA-oleate
HA-linoleate
Mean diameter (nm)
Polydispersity
ꢁ (mV)
80.57 0.27
0.154
−33.2
114.1 0.91
0.188
−25.1

E. Calce et al. / Carbohydrate Polymers 143 (2016) 84–89
89
heating, our results showed that microwave radiations improved
the chemical process, both in terms of yield and also purity
of the final product. In conclusion, taking in consideration the
energy aspects, an already biocompatible chemical process, when
performed under MW-activation, further moved toward “green
chemistry”. In this regard, the MW method employed to induce
the HA-oleate and HA-linoleate self-assembly offered the great
advantage of eliminating the use of organic solvent, which makes
the whole process even more eco-compatible in terms of chemical
consumption.
de la Hoz, A., Diaz-Ortiz, A., & Moreno, A. (2005). Microwaves in organic synthesis.
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Duan, H., Wang, D., & Li, Y. (2015). Green chemistry for nanoparticle synthesis.
Chemical Society Review, 44, 5778–5792.
Ganesh, S., Iyer, A. K., Morrisey, D. V., & Amiji, M. M. (2013). Hyaluronic acid based
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Acknowledgements
The authors thank Mr. Leopoldo Zona (National research Coun-
cil, Naples) for technical assistance in NMR spectroscopy and Dr.
Paola Ringhieri (University of Naples) for helpful insights of the
nano-particle preparation.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carbpol.2016.02.001.
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