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Ultrasonic synthesis of stable oil filled

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microcapsules using thiolated chitosan and their

Cite this: Soft Matter, 2016,
12, 7212 characterization by AFM and numerical simulations
Sinuo Tan,a Srinivas Mettu,*b Matthew Dominic Biviano,b Meifang Zhou,a
Bandar Babgi,c Jonathan White,ad Raymond R. Dagastine*b and
Muthupandian Ashokkumar*a

Received 18th June 2016,
Accepted 29th July 2016
DOI: 10.1039/c6sm01402j
www.rsc.org/softmatter
An experimental protocol has been developed for synthesizing stable core–shell microcapsules using a
biopolymer, chitosan, lacking cross-linkable thiol functional groups. In the first step, thiol moieties were
introduced into the backbone of chitosan using DL-N-acetylhomocysteine thiolactone (AHT). In the
second step, AHT-modified chitosan shelled microcapsules, encapsulating an oil core, were successfully
prepared using high intensity 20 kHz ultrasound. The size of chitosan and AHT modified chitosan
microcapsules was found to be in the range of 1–15 mm. The thickness of the microcapsule shell
increased with an increase in thiol content. The mechanical properties of microcapsules were evaluated
by subjecting the microcapsules to compressive forces by colloidal probe AFM. The stiffness and the
Young’s modulus of the shell of microcapsules were determined by analyzing the force versus indentation
data using Reissner’s theory for indentation of thin elastic shells. The stiffness of AHT modified chitosan
microcapsules was found to be higher than unmodified chitosan microcapsules. The viability of microcapsules
to be embedded into processed food, pharmaceutical and cosmetic products was tested via numerical simu-
lations. The confined capsule in the micro-channel was subjected to linear shear and uniform flows. We
used finite element numerical simulations to determine the deformation of microcapsules in flow as a
function of shear rate and thickness of the shell. The deformation of capsule was found to be linear with
an increase in the shear rate. The deformation decreased with an increase in the thickness of the shell.
Based on the simulations, we predict that the microcapsules would survive processing conditions and
shear rates used in industrial applications.

1 Introduction the release of the substance can be triggered. Encapsulation can

Encapsulation is a technique by which one substance or a
mixture of substances (active or core material) is packaged
within a secondary material (shell or wall material) to form
small capsules. Encapsulation technology is now well accepted
and developed in pharmaceutical, chemical, cosmetics and food
industries.1–3 Application of this technology in the food industry
is of interest since the encapsulated substances can be protected
against moisture, pH, heat and other extreme conditions until
a
School of Chemistry, The University of Melbourne, Parkville, Melbourne,
Victoria 3010, Australia. E-mail: masho@unimelb.edu.au
b
School of Chemical and Biomolecular Engineering, The University of Melbourne,
Parkville, Melbourne, Victoria 3010, Australia. E-mail: smettu@unimelb.edu.au,
rrd@unimelb.edu.au
c
Department of Chemistry, Faculty of Science, King Abdulaziz University,
P.O. Box 80203 Jeddah, Saudi Arabia
d
BIO-21 Institute, The University of Melbourne, 30 Flemington Road, Parkville,
Melbourne, Victoria 3010, Australia
also mask undesired taste or odour.2,4
Several polymeric materials have been used as a shell
material including starch, proteins, lipids and polysaccharides,
etc.2,5–8 Among these materials used in encapsulation, chitosan
(poly-[1 - 4]-b-D-glucosamine) has gained considerable atten-
tion due to its excellent biological and chemical properties such
as biodegradation,9,10 biocompatibility,11 nontoxicity,12 and anti-
bacterial activity.13 There are various methods available to synthe-
size chitosan microcapsules including layer-by-layer assembly
methods,14 membrane emulsification,15 cross-linking techni-
ques,16,17 and sonochemical methods.18,19 Skirtenko et al.,18 for
the first time, prepared oil filled chitosan microcapsules by a
sonochemical process. Compared with other techniques, ultrasonic
synthesis has several advantages that include the ease of operation,
low-cost, shorter reaction time, superior products and no addition
of cross-linking agents, etc.18,20 Despite notable progress reported
in previous studies, the preparation of stable chitosan micro-
capsules as an effective shell material still remains a challenge.

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Suslick et al.21 proposed that the main mechanism for the

formation of proteinaceous microcapsules is the intermolecular
cross-linking from the cysteine oxidation during acoustic cavita-
tion. When the reaction proceeded under an inert atmosphere,
microcapsules were not formed, showing the importance of
disulfide cross-linking on the ultrasonic synthesis of stable micro-
capsules. The intermolecular disulfide cross-linking is believed
to be caused by the hydroperoxyl radicals generated during the
acoustic cavitation.
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Chitosan does not contain free thiol groups and hence

chitosan-shelled microcapsules may be less stable.22 A process
to immobilize thiol moieties into the chitosan backbone has
been conducted mainly by the covalent attachment of –SH groups
using thioglycolic acid, mediated by a water soluble carbo-
diimide. This methodology has some disadvantages:23 first, a
self-coupling reaction between carboxylic acid and thiol groups
may occur to generate thiolester. Secondly urea derivatives as side
products can be generated by the transformation of carbodiimide
during the coupling reaction. Another reagent reported to thiolate
chitosan is 2-iminothiolane (Traut’s reagent).24 However, thiolated
chitosan synthesized by this method showed lower stability.25 The
reason may be the formation of N-substituted-2-iminothiolane,
which caused a decrease in the number of free thiol groups. One
attempt explored to incorporate thiol groups is via nucleophilic
ring-opening reaction with homocysteine thiolactone, catalyzed
by imidazole. Juntapram et al.26 showed that immobilization of
thiolactone on the chitosan backbone can be achieved. In spite of
the addition of imidazole, the conversion of molecules in this
method is 100%.27
In order to strengthen chitosan-shelled microcapsules, we
have explored the possibility of thiolating chitosan by chemical
processes. In this study, we synthesized two batches of thio-
lated chitosan containing various amounts of thiol moieties by
the use of DL-N-acetylhomocysteine thiolactone (AHT). A com-
parison between chitosan and thiol-containing chitosan micro-
capsules in terms of average size, size distribution and shell
thickness of microcapsules has been carried out. The shell
thickness, stiffness and elastic modulus are the key factors that
determine whether the embedded microcapsules survive shear
and processing conditions encountered in the manufacturing
of the final product. Hence, the stiffness and Young’s Modulus
of individual chitosan and modified chitosan shells have been
quantitatively measured and compared using atomic-force micro-
scopy (AFM). Using these modulus data, we carried out numerical
simulations to determine the deformation of microcapsules as a
function of shear rate in flow and thickness of the shell.
2 Experimental
2.1 Materials
Chitosan [low molecular weight, degree of deacetylation (DD)
78% by 1H NMR], DL-N-acetylhomocysteine thiolactone (AHT),
imidazole, lactic acid, tetradecane, acetic acid and Nile red were
obtained from Aldrich Co., USA and used without any purifica-
tion. Acetic acid-d4 (CD3COOD) and deuterium oxide (D2O),
Fig. 1 Schematic representation of the reaction scheme for the synthesis
of AHT modified chitosan.
used as NMR solvents, were purchased from Aldrich Co., USA.
High-purity water was from an inline Millipore RiOs/Origin
System (Milli-Q water). A dialysis kit (Mw cut-off 14 kDa) was
obtained from Aldrich Co., USA. All glassware used in AFM experi-
ments were cleaned using Ajax detergent, soaking in nitric acid,
followed by cleaning in base solution (NaOH), rinsed with Milli-Q
water and dried. Nitric acid was obtained from Ajax fine chemicals
and used without purification.
2.2 Synthesis of AHT modified chitosan
Chitosan was thiolated by covalent attachment using AHT,26
following the reaction scheme shown in Fig. 1. Two batches
of AHT-chitosan (AHT-chitosan 1 and AHT-chitosan 2) were
synthesized by varying the (w/w) ratio of chitosan : AHT and
reaction time. Briefly, an aqueous solution of 1% (w/v) chitosan
in 1% (v/v) lactic acid was prepared in a three-necked flask and
imidazole (0.68 g in 2.5 mL Milli-Q water) was added under
stirring, followed by the drop-wise addition of AHT (1 g and
3 g in 100 mL Milli-Q water). The mixture was stirred for 12 h
and 48 h, respectively, under a nitrogen atmosphere at room
temperature and then purified by dialysis against Milli-Q water.
The products were precipitated with excess acetone, washed
with ethanol and acetone successively, and dried in a vacuum
oven at room temperature.
2.3 Preparation of chitosan and AHT-chitosan microcapsules
A mass of 2.5 mg of chitosan, AHT-chitosan 1 and AHT-
chitosan 2 were dissolved in an aqueous acetic acid solution
(pH 2.88) and then 50 mL of tetradecane was layered at the top
of the solution. A 20 kHz ultrasound generator (Branson) with a

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3 mm diameter horn was used for the microcapsule prepara-
tion.28 The tip of the horn was positioned at the oil–water
interface and the sample was sonicated at an acoustic power of
160 W for 30 s. The temperature of the reaction solution was
maintained at 20 1C. Following microcapsule formation, aqueous
NaOH solution (pH 11) was added to partially neutralize excess
acetic acid. Microcapsules were then collected from the top of the
sonicated solution and washed repeatedly using aqueous acetic
acid solution (pH 4) 5 times.
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2.4 Characterization
2.4.1. 1H nuclear magnetic resonance spectroscopy (NMR).
1
H NMR spectra of chitosan and AHT-chitosan in 1% (v/v)
CD3COOD in D2O were recorded on a Varian MR400 NMR
spectrometer operating at 400 MHz at 25 1C.
2.4.2. Determination of thiol content. Since the character-
ization (e.g. thickness and stability) of microcapsules is depen-
dent on the thiolation level,28 the thiol content of AHT modified
chitosan was evaluated by analyzing the 1H NMR spectra of AHT-
chitosan according to eqn (1).
 
 1
 1  106  ð1=2  IHa Þ
Thiol content mmol g ¼    (1)
M  1=6  IðH2H6Þ
Here, IHa is the integral of the chemical shift of Ha at 2.75 ppm
and IH2–H6 is the integral of the 1H peak between 3.0 and 4.2 ppm.

M is the average molecular weight of the monomer, considering
the degree of deacetylation of chitosan, 78%.
2.4.3. Optical, fluorescence and scanning electron micro-
scopic analyses. Optical microscopy (Olympus Model IX71) was
used to record the images of microcapsules dispersed in water.
Fluorescence microscopy (Olympus Model IX71) was used for
observing tetradecane encapsulated inside microcapsules. Nile
red dissolved in tetradecane was used as a fluorescence probe.
The average size and size distribution of microcapsules were
assessed by measuring over 400 microcapsules using optical
microscopic images. A scanning electron microscope (SEM) (FEI
Quanta) was used to examine the morphology of AHT modified
chitosan microcapsules. The shell thickness of microcapsules
was measured from the cross section of the microcapsule shell
obtained by SEM.29
2.4.4 Sample preparation for atomic force microscopy
(AFM) experiments. Ultrasonically cross-linked chitosan micro-
capsules filled with tetradecane were immobilized on circular
glass substrates as described in detail in our previous paper.30
We briefly describe the procedure here for the sake of com-
pleteness. Immobilization of microcapsules onto a rigid sub-
strate was necessary to carry out AFM indentation experiments.
Four to five drops of concentrated suspension of microcapsules
were added to an acetic acid solution of pH B 4 stored in a
10 mL plastic vial. The capsules slowly cream and float on top
of the solution due to the density difference of tetradecane and
water. More acetic acid solution was added to the vial until it
was filled up to the brim of the vial. The circular glass bottom of
the AFM fluid cell was made hydrophilic by cleaning sequen-
tially for an hour in Ajax, sodium hydroxide and concentrated
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Fig. 2 Schematic of the microcapsule indentation experiments carried
out by AFM using a silica colloidal probe attached to a microcantilever (a).
Optical microscopy images of the capsule immobilized on a glass slide:
chitosan (b), AHT-chitosan 1 (c) and AHT-chitosan 2 (d). The compression
experiments are carried out in a fluid cell filled with aqueous acetic acid
solution at pH B4.
nitric acid. It was then washed with excess Milli-Q water and
dried using ultra-purified nitrogen gas. The dried glass disks
were then cleaned using Ozone (UV/Ozone ProCleaner Plus,
Bioforce Nanosciences) for 20 minutes to remove any residual
organic contaminants. The cleaned glass disks were then
deposited on top of the plastic vial containing microcapsules
and left on the vial for approximately 24 h for the microcapsules
to slowly cream and firmly attach to the glass slide. A typical
optical microscopy image of immobilized microcapsules along
with the colloidal probe used to compress them is shown in
Fig. 2 for various chitosan capsules.
The circular glass disk with immobilized microcapsules was
attached to the AFM fluid cell and 2 mL of pH B4 acetic acid
solution was added to the fluid cell. Some of the capsules that
were loosely anchored to the glass disk were washed off with the
addition of acetic acid solution. The microcapsules that were
firmly anchored remained on the surface and did not move
during the addition of more liquid. The AFM fluid cell was then
washed several times by addition and subsequent removal of
acetic acid solution. This washing procedure was continued until
all the microcapsules that were floating in the solution were
removed from the fluid. We also further tested the anchoring of
microcapsules to the glass bottom of AFM fluid by adding 1 mL of
acetic acid solution to the fluid cell while the microcapsules were
observed using an inverted microscope (40 inverted microscope
objective, Nikon Eclipse Ti-U).
2.4.5 AFM indentation experiments. We prepared the silica
colloidal probe used for indentation experiments (Fig. 2) using
a method described in Ducker et al.31 The diameter of the
colloidal probe (silica particle) was 19.5 mm. The spring constant
of the probe was measured by the method of Hutter–Bechhoefer32
and it was 0.1 N m1  0.02 N m1. The AFM (MFP-3D, Asylum
Research, Ca, USA) head loaded with the silica colloidal probe
was slowly lowered into the fluid cell containing microcapsules
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in pH B4 acetic acid solution. The sensitivity (deflection/applied

voltage) of the colloidal probe was measured by recording a
force curve between the bare glass substrate of the fluid cell and
the colloidal probe until a constant compliance region was
achieved. The constant compliant region was used to calibrate
the optical lever sensitivity of the probe, i.e. the extent to which the
probe deflects for a given displacement of the peizoactuator. This
measurement was used as a reference for a rigid substrate. The
individual microcapsules of appropriate size (Z3 mm) were
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located and the diameter was measured by recording an optical

image using an inverted microscope (40 objective, Nikon Eclipse
Ti-U). The colloidal probe was then engaged and slowly lowered
onto the microcapsule until a finite deflection of the probe
was recorded. The vertical alignment of the colloidal probe
with that of the capsules was achieved further by adjusting the
position of the fluid cell (hence microcapsule) with a micro-
manipulation stage.
We restricted the velocity of indentation to 100 nm s1 in
order to eliminate hydrodynamic effects resulting from the flow
of liquid based on the viscosity of acetic acid solution. The
maximum force of indentation was fixed to approximately
10 nN. The indentation experiment was repeated 10 times on
each microcapsule particle to check the reproducibility of the Fig. 3 Representative 1H NMR spectra of (a) chitosan, (b) AHT-chitosan,
measurement. The size of the capsules does not change with the and (c) magnification in the vicinity of 2.15–2.30 ppm. H2O was used as a
reference (4.8 ppm). The signal at 2.20 is attributed to CH3 protons in the
application of 10 nN load. For each sample type, 10 different
reaction solvent acetone.
individual microcapsules were located on the glass surface and
the indentation experiment was repeated on each of them. The
force versus indentation data obtained on the microcapsules was protons of the chitosan backbone. After grafting of AHT onto
analyzed using Reissner’s theory33 as described in detail in later the chitosan, new signals that appeared at 2.75 and 2.26 ppm
part of the article. Some adhesion was observed between the which were assigned to Ha and Hb of AHT-chitosan demon-
silica particle and microcapsules in the retraction portion of strated the successful synthesis of AHT-chitosan. Perhaps due
the force distance curve. Thus, for the analysis of the elastic to being part of a polymer, there was no evidence for splitting
properties of the microcapsules, only the approach (indentation) from spin–spin coupling between Ha and Hb. The appearance of
portion of the force–distance curve was used to estimate the the shoulder on the acetyl signal at ca. 2.0 ppm was also consistent
Young’s modulus. with the incorporation of AHT. The signal at 2.20 ppm was
attributed to the reaction solvent acetone which got trapped within
the polymer. The thiol contents for AHT-chitosan 1 and AHT-
3. Results and discussion chitosan 2, as calculated from eqn (1), were 176 and 235 mmol g1,
3.1 Synthesis of AHT-chitosan respectively.

In this work, DL-N-acetylhomocysteine thiolactone (AHT) was used
for the incorporation of thiol groups into the chitosan backbone
to increase the shell strength of chitosan-based microcapsules.
This involved the preformation of the imidazole activated inter-
mediate, HS–CH2–CH2–CH(NHCOCH3)–C(O)–Im (where, Im =
imidazole),26 followed by acylation of the chitosan amino groups
and formation of amide bonds.
1
3.2 H nuclear magnetic resonance spectroscopy (NMR)
The aim of using NMR spectroscopy is to establish the extent of
thiolation of the chitosan. 1H NMR spectra of chitosan and
AHT-chitosan are shown in Fig. 3. The signal at 2.07 ppm can
be attributed to the three protons in the acetyl group of N-acetyl
glucosamine (GlcNAc), whilst that at 3.17 ppm represents the
H2 proton of the glucosamine (GlcN) residues. The chemical
shifts between 3.50 and 4.30 ppm correspond to the H3–H6
3.3 Synthesis of chitosan and AHT-chitosan microcapsules
When tetradecane was layered on the surface of AHT-chitosan
aqueous solution and sonicated as described in the Experi-
mental section, stable AHT modified chitosan microcapsules
were formed (Fig. 4(b) and (c)).
The AHT modified chitosan microcapsules showed a rough
surface morphology as observed by scanning electron micro-
scopic analysis (Fig. 5). However, chitosan microcapsules could
not be found by SEM since they were broken under the vacuum
environment of SEM due to a weaker shell. In order to confirm
the encapsulation of tetradecane, Nile red, a fluorescent probe,
was dissolved in tetradecane prior to encapsulation. The fluores-
cence optical microscopic images (Fig. 4(e) and (f)) indicated that
AHT-chitosan microcapsules were filled with tetradecane con-
taining Nile red. The mechanism for the formation of AHT
modified chitosan microcapsules is similar to that reported in

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Fig. 4 Optical and fluorescence microscopic images of the micro-
capsules of chitosan (a) and (d), AHT-chitosan 1 (b) and (e), and AHT-
chitosan 2 (c) and (f).
Fig. 5 SEM images of AHT modified microcapsules: (a) shell of collapsed
capsules and (b) intact microcapsule.
previous studies.34 In brief, it involves three processes: emulsi-
fication of the non-aqueous phase to form a suspension of
microdroplets in an aqueous solution, aggregation of AHT-
chitosan at the oil–water interface and disulfide cross-linking
between AHT-chitosan molecules caused by the hydroperoxyl
radicals generated during the acoustic cavitation process.
The sizes of chitosan, AHT-chitosan 1 and AHT-chitosan 2
microcapsules were found to be similar (Table 1) in the range of
1–15 mm (Fig. 6). The thickness of microcapsules increased with
an increase of the thiol content (Table 1). The formation of
thicker shelled microcapsules may be due to an increase in
hydrophobic interaction between AHT-chitosan molecules35 at the
oil/solution interface. Also, amidation of chitosan can decrease
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Table 1 Average size, thickness, stiffness and modulus of chitosan, AHT-
chitosan 1 and AHT-chitosan 2 microcapsules
Average Thickness/ Average stiffness/ Average
Samples size/mm nm (mN m1) modulus/MPa
Chitosan 4.5  0.2 148  2 24.0  0.57 0.83  0.02
AHT-chitosan 1 4.3  0.1 165  5 52.9  2.78 1.44  0.08
AHT-chitosan 2 4.0  0.1 180  3 44.3  1.92 1.01  0.05
Fig. 6 Size distributions of chitosan, AHT-chitosan 1 and AHT-chitosan 2
microcapsules measured using an optical microscope.
the intermolecular electron repulsion, which could increase the
polymer aggregation at the interface.
3.4 AFM indentation results
In Fig. 7, we show the approach branch of a typical force versus
indentation curve obtained during the indentation of chitosan
microcapsules (diameter B 4 mm) by a silica colloidal probe.
The nearly vertical black filled circles shown in the figure is the
force versus indentation curve against a rigid glass surface.
No indentation was observed into the glass reference surface at
these applied loads. In the case of microcapsules, which are
deformable, indentation into microcapsules increases with an
increase in applied load. The red solid filled circles correspond
to chitosan microcapsules whereas green and blue filled circles
correspond to AHT modified chitosan microcapsules. We show
force versus indentation curves on five different individual
capsules for each type of the chitosan sample. As observed,
the force curves are reproducible across the capsules of similar
size for the same chitosan sample type. The solid lines show the
approximate average slope of force curves for each chitosan
sample. The microcapsules exhibited linear elastic behaviour,
i.e. the indentation increased approximately linearly with an
increase in applied load. We did not observe any instabilities
such as buckling or inflection of shell as we restricted the
applied load to less than 10 nN. According to the theory of thin
shell indentation,33 in the small deformation regime (indentation–
shell thickness), the slope of the force versus indentation curve is
proportional to the stiffness of the microcapsule. The modulus of
the shell and the resistance of the microcapsule to deformation
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Fig. 7 Experimental force curves for the indentation of chitosan micro-
capsules with and without SH modification. Example force curves on five
different capsules for each type of chitosan are shown as red, green and
blue solid filled circles. The black filled circles represent the force versus
indentation curve against a glass surface given as a rigid reference sub-
strate. The red, green and blue solid lines indicate the approximate slopes
of the force curves that are called the stiffness of the capsule given by
4Es
Ks ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffih2 .
R 3ð 1  n 2 Þ
from external applied loads are proportional to the stiffness of
the shell. Higher shell stiffness indicates stronger microcapsules.
The slope of the force curve for AHT modified chitosan micro-
capsules is higher than unmodified chitosan samples (Fig. 7).
This indicates that modified chitosan microcapsules are stronger
than the unmodified microcapsules.
In order to determine the quantitative effects of AHT modi-
fication on stiffness and modulus, force versus indentation data
were analyzed by using Reissner’s theory.33 When a spherical
microcapsule of radius R and shell thickness h is compressed by
a rigid sphere of radius R1 at a given load F, the indentation (d) of
the microcapsule is related to applied load33 by
F = Ksd (2)
Here, F and d are applied load and indentation respectively.
Ks represents the microsphere stiffness given by:
4Es
Ks ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffih2 (3)
R 3ð1  n 2 Þ
where Es, h, and n are Young’s modulus, shell thickness, and
the Poisson’s ratio of the microspheres respectively. Since the
silica particle is rigid (E1 B 68 GPa) compared to microcapsules
(Es B 1–2 MPa), its deformation is neglected.
In Fig. 8, we show the shell stiffness and Young’s modulus as
a function of the diameter of the microcapsules. The stiffness of
microcapsules did not vary strongly with the size of capsules
within the range of diameters used in the experiments. The
stiffness of AHT modified chitosan varied between 40 and
60 mN m1 whereas they were between 20 and 30 mN m1
for unmodified chitosan. We compare the average stiffness and
Young’s modulus of AHT modified and unmodified chitosan
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Fig. 8 Stiffness (Ks) (a) and Young’s modulus (Es) (b) of chitosan, AHT-
chitosan 1 and AHT-chitosan 2 microcapsules as a function of micro-
capsule diameter. The modulus is calculated from the stiffness of the shell
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
R 3ð 1  n 2 Þ
using the relation Es ¼ Ks .
4h2
microcapsules in Table 1. The stiffness of AHT modified chitosan
microcapsules was almost twice that of unmodified chitosan.
The Young’s modulus of AHT modified chitosan microcapsules
(B1–1.44 MPa) was higher than the modulus of unmodified
chitosan (B0.83 MPa). It should be highlighted that thiol
contents for AHT-chitosan 1 and AHT-chitosan 2 are 176 and
235 mmol g1, respectively. This corresponds to the extent of
thiolation of about 3% and 4%, respectively. The difference in
the extent of thiolation between these samples is insignificant
within experimental error. The measured stiffness values for
thiolated capsules were 52.9  2.78 and 44.3  1.92 mN m1.
The minimum value for stiffness of AHT-chitosan 1 taking the
standard deviation in the measurement into consideration is
50.12 mN m1 whereas the maximum value for AHT-chitosan 2
is 46.22 mN m1. The change in the stiffness for two thiolated
chitosan samples is very small compared to an increase in stiffness
from non-thiolated chitosan (24  0.57 mN m1) to thiolated
chitosan microcapsules (52.9  2.78 and 44.3  1.92 mN m1).
Based on the small change in stiffness between two thiolated
chitosan samples, we expect that their overall strength is approxi-
mately the same. In order to observe a significant increase in
strength with an increase in thiolation, the extent of modification
should be significantly higher (more than 10%).28
3.5 Deformation of microcapsules in shear and uniform flows
In order to use the chitosan microcapsules to encapsulate
drugs, fragrances and nutrients, capsules should survive shear
forces encountered during the processing of final products into
which they are embedded. The microcapsules indeed deform in
shear flows, but the extent of deformation depends on shell
thickness and elastic modulus. Hence, it is useful to estimate
the response of microcapsules by subjecting them to shear
flows by varying the shear rate (velocity). We use the modulus
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data of the AFM with numerical experiments to predict the
deformation of microcapsules and the resulting equilibrium
shapes in linear shear and uniform flows.
The deformation of a freely suspended capsule with a thin
elastic shell subjected to uniform flows, shear and extensional
flows, has been well studied in the literature.36–51 First theo-
retical studies on capsule deformation were carried out by
Barthes-Biesel et al.36–44 whereas first experiments were carried
out by Chang and Olbricht45,46 as well as interesting work by
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Fischer on the deformation of protein coated droplets under
shear.52,53 These studies focused on the effect of elastic modulus,
shear rates, viscosity ratios, membrane pre-stress and membrane
constitutive laws on the deformation of a freely suspended
capsule in symmetric shear flow. Buckling,40,42 wrinkling49–51
and folding47 of the membrane and breakup45,49 of the capsules
with an increase in shear rates have been observed. The reader is
referred to a detailed review by Biesel41 for more information
on this topic.
In our approach, we used finite element multiphysics simu-
lation software COMSOLs to carry the numerical calculations.
We used the fluid-structure interaction (FSI) module in
COMSOLs that combines fluid flow with solid mechanics to
simulate the interaction of fluid with a deforming structure and
vice versa.
3.5.1 Geometry and mesh. We show the geometry used in
the simulations in Fig. 9(a) and (b) for linear shear and uniform
flows respectively. A 2 mm radius chitosan microcapsule filled
with tetradecane is fixed to the bottom wall of a rectangular
channel of 30 mm length and 15 mm width. The microcapsule is
surrounded by water. The microcapsule is then subjected to
steady-state linear shear or uniform flows as schematically
Fig. 9 Geometry used in COMSOLs for simulation of deformation of
microcapsules in linear shear (a) and uniform flow (b). The microcapsule is
surrounded by water. The un-deformed radius of the microcapsule is
2 mm. The capsule is fixed to the bottom wall of the channel. The fine mesh
around the capsule (c).
7218 | Soft Matter, 2016, 12, 7212--7222
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Paper
shown in Fig. 9(a) and (b). Typical fine mesh around the
microcapsule used for simulations is shown in Fig. 9(c).
3.5.2 Equations and boundary conditions
(a) Steady state equations for fluid flow and solid deformation
in 2-dimensions. Two dimensional Navier–Stokes equations for
fluid flow is given by
r(Vr)V = rG (4)
where
G = [pI + m(rV + (rV)T)] (5)
r, m, p and V are density, viscosity, pressure and velocity of fluid.
The continuity equation is given by
r(rV) = 0 (6)
The equation for solid deformation (shell of the microcapsule)
in 2D is given by
rs = FV (7)
where s is the stress on the solid (2D) and FV is the volumetric
body force acting on the solid. FV is set to zero as there is no
external body force acting on the microcapsule. The shell of the
microcapsule is treated as an isotropic linear elastic material
where shear stress is linearly proportional to strain.
s B Es e (8)
where Es is the Young’s modulus. Poisson’s ratio is assumed to
be 0.49.
(b) Boundary conditions. At the inlet boundary (Fig. 9(a) and (b)),
the normal inflow velocity boundary condition is used where the
velocity profile is given by V(y) = V0(y/w) for linear shear flow and
V(y) = V0 for uniform flow. At the outlet boundary, the outflow
condition is used with no viscous stress. This boundary condition
ensures that the presence of the boundary does not affect the
solution inside the channel. On both the top and bottom walls the
symmetry boundary condition is used where shear stress and
normal flow are zero. The capsule was fixed to the bottom wall
by the fixed constraint boundary condition at the bottom boundary
of the capsule. At the liquid–solid interface (outside and inside the
microcapsule) the stress continuity boundary condition is used
where the stress due to fluid flow is transferred to solid boundary
and vice versa. This results in coupled deformation of the micro-
capsule due to fluid flow and flow developing due to the deforma-
tion of the microcapsule.
V = usolid (9)
Normal stress transfer at the fluid solid interface boundary
is given by
sn = Gn (10)
where G is the shear stress due to fluid flow given by eqn (5).
For more details on the equations and boundary conditions,
the reader is referred to the user manual of the COMSOLs
Fluid Structure Interaction (FSIs) module.
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3.5.3 Deformation in linear shear and uniform flows. The
microcapsule deforms in shear flow due to viscous forces acting
on the capsule wall but there is a resistance to deformation
resulting from the elastic nature of the shell of the microcapsule.
The non-dimensional number that controls the microcapsule
deformation is the elasto-capillary number which is the ratio of
viscous forces to elastic resistive forces and is given by
m_gR
CaE ¼ (11)
Es h
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of shear flow that corresponds to shear rates (dV/dx B V0/w) of
166 to 1333 s1, approximately. The velocity of approach in the case
of uniform flow was varied from 0.25 to 2 cm s1. We restricted the
velocities to very low values (o2 cm s1) as we treated the shell of
the microcapsule as an incompressible linear elastic material. The
microcapsule follows volume incompressibility as the deformation is
restricted to the linear range. As indicated by Biesel,41 if the capsule
undergoes large deformations, the membranes of the capsule get
stretched and hence different constitutive laws for membrane

stretching needs to be used. In order to simulate the increase in

where m, g_ and R are the viscosity of the surrounding fluid, the the thickness of chitosan microcapsules with AHT modification, we
shear rate and the radius of the microcapsule respectively. varied the shell thickness from 150 nm to 180 nm keeping the radius
Es and h are the Young’s modulus and thickness of the shell of capsules constant to 2 mm. We fixed the Young’s modulus of the
of the microcapsule. The elasto-capillary number is alternatively shell to 1 MPa in order to study only the effects of varying the shell
referred to as the dimensionless shear rate. For a given shell thickness and shear rate on capsule deformation. This modulus
thickness and modulus, the capillary number increases with an corresponds to experimentally measured Young’s modulus of
increase in the shear rate. The deformation of a freely suspended chitosan shells by AFM (Table 1). We carried out steady state
capsule in symmetric shear flow is generally defined by Taylor’s simulations using COMSOLs and calculated equilibrium shapes
deformation parameter given by of the microcapsule as a function of velocity. Numerical simula-
LB tions in 2D instead of 3D were conducted even though capsules
D¼ (12)
LþB deform in 3D in the experiments. However, Breyiannis and
Pozrikidis55 have shown that for small deformations, 2D deforma-
where L and B are the major and minor axes of the deformed capsule
tion corresponds very well to 3D deformation. In order to save
that assumes an ellipsoidal shape in linear shear flow. Biesel44 has
computational time, we restricted our simulations to 2D.
shown that for a purely elastic shell in the small deformation
We show the equilibrium deformed shapes of the microcap-
regime Taylor’s deformation parameter is linearly proportional
sule as a function of velocity and shell thickness in Fig. 10 and 11
to the elasto-capillary number by the following relation.49
25
D¼ CaE (13)
4
Koleva and Rehage49 showed that polysiloxane microcapsules
follow such a linear deformation in shear flow until they break up
when CaE 4 0.003. Though the deformation of a freely suspended
capsule is an interesting phenomenon, in practical applications
where capsules are sheared under processing conditions the
capsules may not be freely suspended and symmetrically sheared.
However, the capsules may be attached to solids in the processing
mixture that is being sheared and hence they resemble a tethered
capsule instead of a freely suspended capsule. In this case, the
geometry of shear flow resembles that geometry we used in the
current simulations shown in Fig. 9(a) and (b). There is one
computational study by Berry et al.54 who studied the deformation
of tethered leukocytes and platelets in linear shear flows. In their
numerical study spherical and non-spherical platelets were tethered
to the blood vessel wall and their deformation in linear shear flow
was studied as a function of the shear rate. Berry et al.54 assumed an
infinitely thin shell for the platelets and showed that the relative
deformation of tethered capsules was linear as a function of CaE up
to 0.25. In our simulations, as the microcapsule is not symmetrically
sheared, Taylor’s deformation parameter may not be applicable.
Instead, we define the deformation of the capsule by maximum
relative deformation relative to its undeformed radius given by
Fig. 10 Equilibrium shapes of deformed microcapsules in linear shear
DXmax flow as a function of velocity. Equilibrium undeformed shapes of micro-
Dmax ¼ (14)
R0 capsules are shown by black circles. Vertical panels (a) and (b) correspond
to microcapsules with shell thickness of 150 and 180 nm respectively.
In the numerical simulations, the maximum velocity of Horizontal panels (1), (2) and (3) correspond to velocities of 0.5, 1.5 and
approach of fluid is varied from 0.25 to 2 cm s1 in the case 2 cm s1, respectively.

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Soft Matter Paper

for linear shear and uniform flows, respectively. The unde-

formed equilibrium shape of the capsule is shown by a black
circle in each figure. The color scale indicates the extent of
deformation where the blue color corresponds to low values and
dark red corresponds to high values. For a given thickness of the
shell, the deformation increases with an increase in velocity both
for the linear shear and uniform flows (Fig. 10 and 11 (1), (2)
and (3)). For a given velocity, the deformation of the capsule
decreases with an increase in the thickness of the shell from
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150 nm to 180 nm (Fig. 10 (3) and 11 (3)). We summarize the

effect of the increase in the velocity of the flow and thickness of
the shell on the maximum relative deformation of the micro-
capsule in Fig. 12(b) and (c) for linear shear and uniform flows
respectively. The deformation decreases with an increase in
thickness for both linear shear and uniform flows. The deforma-
tion is more in uniform flow compared to shear flow for the
same velocity of approach. Shear rates corresponding to the velo-
cities are shown in Fig. 12(b) for linear shear flow. As observed,
in the small deformation regime where velocities o2 cm s1,
and shear rates o1333 s1, the deformation of capsules is linear
with the shear rate. An interesting observation is that even at a
very high shear rate of 1333 s1, the microcapsule did not
break. Hence, our simulations show that the microcapsules
would deform without breaking in the processing conditions
encountered in the manufacturing of pharmaceutical, food and
fragrance products with embedded microcapsules.

Fig. 12 Maximum relative deformation (DXmax/R0) of microcapsules as a

function of velocity (shear rate) for linear shear flow (a) and uniform flow
(b) obtained from numerical simulations. Red, blue and green filled circles
correspond to microcapsules with shell thicknesses of 150, 165 and 180 nm.
The solid lines are shown as a guide to the eye.

Fig. 11 Equilibrium shapes of deformed microcapsules in uniform flow as
a function of velocity. Equilibrium undeformed shapes of microcapsules
are shown by black circles. Vertical panels (a) and (b) correspond to
microcapsules with shell thicknesses of 150 and 180 nm respectively.
Horizontal panels (1), (2) and (3) correspond to velocities of 0.5, 1.5 and
2 cm s1, respectively.
4 Conclusions
Thiolated chitosan has been successfully synthesized by
covalent attachment of AHT onto the chitosan backbone.
Unmodified and AHT-modified chitosan shelled microcapsules
were ultrasonically prepared. The microcapsules showed a
rough shell morphology. Using a fluorescent probe, the encap-
sulation of oil inside the AHT modified microcapsules has been
demonstrated. There was no significant difference in size and
size distribution between chitosan microcapsules and AHT
modified chitosan microcapsules. We showed that the shell
thickness of microcapsules can be controlled by the incorpora-
tion of thiol groups. The thickness of microcapsules increased
with an increase of the thiol content. The mechanical strength
of the shell of the microcapsules was evaluated using AFM. The
stiffness of the AHT modified chitosan shell was found to be
almost twice that of unmodified chitosan. The Young’s modulus of

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Paper
AHT modified chitosan microcapsules ranged from 1 to 1.44 MPa
whereas it was about 0.8 MPa for unmodified chitosan micro-
capsules. The deformation of microcapsules in linear shear and
uniform flows was determined using finite element numerical
simulations using the modulus data from the AFM data. The
equilibrium shapes of deformed capsules were determined as a
function of the shear rate of the flow and shell thickness of the
microcapsule. The deformation of the microcapsule increased
linearly with increasing shear rate whereas it decreased with
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an increase in shell thickness. The simulations suggest that the
capsules would survive process conditions and shear rates used
in the manufacturing of final products.
Acknowledgements
The authors thank the Australian Research Council for the
award of an ARC-DP grant.
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