Polymer 54 (2013) 4972e4979

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Polymer
journal homepage: www.elsevier.com/locate/polymer

Microfluidic self-assembly of polymeric nanoparticles with tunable

compactness for controlled drug delivery
Erfan Dashtimoghadam a, Hamid Mirzadeh a, *, Faramarz Afshar Taromi a, Bo Nyström b
a
Department of Polymer Engineering and Color Technology, Amirkabir University of Technology, 424 Hafez Avenue, Tehran, Iran
b
Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, N-0315 Oslo, Norway

a r t i c l e i n f o a b s t r a c t

Article history: A central challenge in the development of polymeric nanoparticles for various applications is precise
Received 4 May 2013 engineering of desired physicochemical characteristics in a reproducible manner. The present work
Received in revised form concerns the use of microfluidics to control the local polymer concentration inside polymeric nano-
2 July 2013
particles. It is demonstrated that the compactness of nanoparticles based on self-assembled hydro-
Accepted 4 July 2013
Available online 13 July 2013
phobically modified chitosan (HMCs) biopolymer can be dictated with tunable rapid mixing via
hydrodynamic flow focusing in microfluidic channels. It is shown by varying the flow rates, as well as the
hydrophobicity of the chitosan chains that the self-assembly behavior of the chains can be controlled by
Keywords:
Microfluidic
optimizing the size and compactness of the species, along with a more narrow size distribution of the
Self-assembly nanoparticles. The size of the particles increased with increasing mixing time, whereas smaller and more
Nanoparticle compact nanoparticles, comprising of a less number of aggregated chains, are produced for chitosan at
higher degrees of hydrophobicity. It was realized that at higher degrees of hydrophobicity and at mixing
times longer than the time of aggregation, nanoparticles comprising of almost the same number of
hydrophobic stickers were formed. Furthermore, we explored the effectiveness of microfluidic directed
to assemble HMCs and to encapsulate paclitaxel (PTX), a common anticancer drug, which revealed
remarkably higher encapsulation efficiency compared to the conventional bulk method. The in-vitro
release of PTX from the prepared nanoparticles was evaluated to investigate the effect of compactness of
the particles on the release profile. The estimated values of the diffusion coefficient of PTX up to 50%
release implied controlled sustainability of the drug release with respect to the compactness of the
nanoparticles, and a remarkable improvement compared to the uneven bulk mixing method. These
results indicate a high potential of the microfluidic approach for precise bottom-up controlling physi-
cochemical properties of polymeric nanoparticles for various applications, such as controlled drug de-
livery systems.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction and improving stability of the labile agents from degradation,

Recent advances of nanotechnology in medicine, termed nano-
medicine, have offered fundamental new capabilities for evolution
of pharmaceutics and biotechnology [1]. Over the past years,
devoted efforts to develop smart nanocarriers have provided
great hopes for various biomedical applications [2,3]. Polymeric
nanoparticle delivery systems offer many distinctive advantages,
such as increasing bioavailability of poorly soluble drugs, ability
to passively accumulate the drug at the tumor site through the
enhanced permeation and retention, providing targeted delivery
* Corresponding author.
E-mail address: mirzadeh@aut.ac.ir (H. Mirzadeh).
thus extending the effectiveness of the therapeutically active
agents [4,5].
It is well-known that crucial physicochemical properties of
nanoparticles, including size, morphology, compactness and sur-
face functionality profoundly affect the particles biodistribution,
release profiles, and mucoadhesion [6,7]. In this regard, an impor-
tant challenge in the design of polymeric nanoparticles is to control
the mixing processes required for the synthesis to proceed in a
reproducible way, which is critical for identification of optimal
characteristics for any desired application [8]. However, inherent
lack of control over mixing in the conventional bulk synthesis
methods, typically results in the production of poorly defined
nanoparticles with deteriorated physicochemical properties; this
may hamper the particles to reach clinical trials [9,10].

0032-3861/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.polymer.2013.07.022
 E. Dashtimoghadam et al. / Polymer 54 (2013) 4972e4979
Microfluidics has emerged as an innovative and distinct
approach for manipulation of extremely small amount of reagents,
providing precise control of mixing and physical processes at mi-
croscales [11e13]. Recently, the continuous flow microfluidic
method has been demonstrated to be a promising platform for
synthesis of various nanostructures, such as polymeric complexes,
liposome-hydrogel hybrid particles, block copolymer micelles,
and homogenous nanoparticles [14e19]. Specifically, microfluidics
has shown to be an efficient bottom-up technique for synthesizing
nanoparticles with excellent control over composition, morphology,
size, and size distribution [20,21].
Among various biopolymers, chitosan is one of the most
promising biomacromolecules for preparation of nanoparticles
with different characteristics for biomedical and pharmaceutical
applications [22,23]. The considerable attention towards chitosan is
owing to its outstanding biologic properties, including biodegrad-
ability, biocompatibility, mucoadhesivity, bioactivity, and its ca-
pacity to open tight junctions [24e27]. Amphiphilic derivatives of
chitosan have shown ability to self-assemble into nanoparticles
independent of ionic gelation or chemical crosslinking [28,29]; this
feature facilitates encapsulation of drugs under gentle conditions
without harsh formulation processes [30].
The objective of the present study is to demonstrate the po-
tential of microfluidic directed synthesis of well-defined nano-
particles, based on self-assembly of hydrophobically-modified
chitosan chains, with respect to drug delivery application. A special
focus of the current work is to understand the effect of controlled
self-assemble of polymeric chains on the size and compactness of
the resultant nanoparticles. As the compactness significantly affects
physicochemical quantities of the nanoparticle delivery systems, a
special emphasis will be placed on its influence of, e.g., degree of
swelling, drug release rate, deformability, circulation time, stability,
and agglomeration of polymeric nanoparticles prepared from chi-
tosan of different hydrophobicity. All these factors have been
shown to intimately rely on the compactness of the particles
[2,6,31,32]. Hence, current research is quantitatively dealing with
the intricate interplay between the mixing time of self-assembled
chitosan chains and the compactness of the nanoparticles. Finally,
the encapsulation efficiency, release pattern, and diffusion coeffi-
cient of the drug paclitaxel from prepared nanoparticles will be
investigated.
2. Experimental
2.1. Materials
Chitosan with a deacetylation degree of 84% was acquired
from Pronova Biopolymers. Size-exclusion chromatography mea-
surements revealed a weight-average molecular weight (Mw)
of w4
l05 and a polydispersity index (Mw/Mn) of w2.7. The hy-
drophobic modification of chitosan was accomplished through
the reaction of amino groups on the biopolymer chains with a
C12-aldehyde. The substitution of C12 aliphatic groups on the chi-
tosan backbone was confirmed by 1H NMR spectroscopy (Bruker
400 MHz). The details of the modification procedure and charac-
terization methods have been described elsewhere [33,34].
The substitution degree of the hydrophobically modified
chitosan (HMCs) samples was determined employing the
ninhydrin method [30]. Briefly described, HMCs samples were
dissolved in an aqueous acetic acid solution, followed by adding
0.5 mL of 4 M acetic acid/acetate buffer (pH ¼ 5.5) into 0.5 mL of
the prepared solutions. Subsequently, 1 mL of ninhydrin reagent
(SigmaeAldrich) was added and the test tubes were placed in a
boiling water bath for 20 min. The solutions were then cooled to
room temperature and the absorbance was read at 570 nm
4973
(Shimadzu UVmini-1240 UVeVis spectrophotometer). The un-
modified chitosan was used as the control sample and the acetic
acid/acetate buffer solution was used as the blank. By using the
ninhydrin assay, the degrees of hydrophobic substitution of the
HMCs samples were determined to be 3.2, 6.3 and 11.1% for
the samples designated as HMCs-1, HMCs-2 and HMCs-3,
respectively.
2.2. Microfluidic device fabrication
The microfluidic device consists of perpendicularly crossed
channels (cross-junction) providing hydrodynamic flow of an
aqueous HMCs solution in a stream of alkaline water (pH 9). A
poly(dimethylsiloxane) (PDMS) device was fabricated according
to a procedure described previously [35,36]. To make the master
molds, SU-8 50 photocurable epoxy was spin-coated on silicon
wafers to a thickness of 60 mm. Baking and lithography pro-
cedures were then performed to create negatively charged
microchannels on the wafer. Subsequently, the wafers were
annealed at 150  C to eliminate superficial cracks. The annealed
mold was coated with a self-assembled monolayer (SAM) of tri-
methylethoxy silane by vapor exposure for 40 min to prevent
sticking of the PDMS to the mold. PDMS (Sylgrad 184) monomer
and curing agent were mixed in 10:1 weight ratio, poured over
the mold, degassed and then cured at 80  C for 1 h. In-/outlet
holes were punched into the cured PDMS using a 150 mm diam-
eter punch, and then the PDMS layer was bound to a glass slide
by means of oxygen plasma (100 mW) for 1 min. The microfluidic
device was composed of three inlets, two for alkaline water and
one for the HMCs solution, and one outlet for the self-assembled
nanoparticles. The designed microchannels were 60 mm high,
150 mm wide, and 1 cm long. The mixing time, smix, was esti-
mated based on Eq. (1) [20],
w2f w2 1
smix w z (1)
4D 9D ð1 þ 1=FRÞ2
where wf, D, w, and FR are the width of the focused stream, diffu-
sivity of the solvent, microchannel width, and the flow ratio of the
HMCs solution stream to the total flow rate of the basic water,
respectively. According to Eq. (1), the diffusive mixing time for
FR ¼ 0.03e0.2 (based on core flow of 0.5 ml min1 and sheath flow
of 16e2.5 ml min1) in this study was estimated to be in the range of
2.5e75 ms.
2.3. Synthesis of HMCs nanoparticles
Nanoparticles were synthesized using the designed micro-
fluidic device through rapidly mixing of HMCs solutions and
basic water in a controlled self-assembly process. In hydrody-
namic flow focusing, the core stream of individually dissolved
HMCs (0.1 wt%) samples is mixed along the two adjacent sheath
streams flowing at higher flow rates (Fig. 1a). At low Reynolds
numbers, the central stream is focused and enables rapid mixing
through diffusion.
For the sake of comparison, a conventional bulk synthesis
method was also utilized to prepare HMCs nanoparticles to mimic
the slow mixing regime. Briefly, a few drops of 1 N NaOH solution
were added to HMCs samples dissolved in 1% aqueous acetic acid
solution under magnetic stirring at room temperature. The basic
principle here is that the induced pH change causes the polymer to
precipitate, but the stirring rate prevents macroscopic phase sep-
aration and particles are formed.
4974 E. Dashtimoghadam et al. / Polymer 54 (2013) 4972e4979
Fig. 1. (a) Schematic illustration of the cross-junction microfluidic system used for
synthesis of HMCs-based nanoparticles; the HMCs solution stream is hydrodynami-
cally focused with lateral sheath flow of basic water (pH ¼ 9). (b) Fluorescence image
of Rhodamine B stream, which is hydrodynamically focused with fluorescein sodium
(scale bar 100 mm). (c) AFM image of HMCs-3 nanoparticles synthesized at flow ratio of
0.031 (scale bar 200 nm).
2.4. Characterization of HMCs nanoparticles
The size and size distribution of the prepared nanoparticles at
pH 7.4 were investigated using dynamic light scattering (Zetasizer
3000HS, Malvern Instruments Ltd., Worcestershire, UK).
Atomic force microscopy (AFM; Bruker’s Dimension FastScan)
and transmission electron microscopy (TEM; CM200-FEG-Philips)
were employed to characterize the shape and size of the pre-
pared nanoparticles. For TEM, dilute suspensions of the nano-
particle samples were deposited onto the Cu grid with a carbon
film, and the instrument was operating at 100 kV accelerating
voltage. ImageJ software was used to analyze the obtained images.
2.4.1. Turbidimetry
The transmittance of the HMCs nanoparticle suspensions
was measured using spectrophotometry (Shimadzu UVmini-1240
UVeVis spectrophotometer) at room temperature. The turbidity
(s) was calculated from the transmittance through the Beere
Lambert law [31],


1 I
s ¼  ln
L I0
where L, I, and I0, are the thickness of the cell, the intensity of the
light transmitted through the sample, and the intensity of the light
transmitted through the solvent, respectively. Measurements were
conducted at 635 nm in at least triplicate, and the mean values are
reported.
2.4.2. Refractive index
The refractive index (n0 ¼ 1.332) of 1% v/v aqueous acetic acid
solution as the solvent was measured with a PTR 46 refractometer
(Index Instruments, UK). The values of the refractive index incre-
ment (dn/dc) of the HMCs solutions were determined with the aid
of the asymmetric flow field-flow fractionation (AFFFF) method,
which was conducted on the AF2000 FOCUS system (Postnova
Analytics, Landsberg, Germany). The values of dn/dc were
measured to be 0.173, 0.180 and 0.185 ml g1 for 1% v/v solutions of
HMCs-1, HMCs-2 and HMCs-3, respectively, at a wavelength of
635 nm.
2.5. Drug encapsulation and in vitro release
Paclitaxel (PTX), a common anticancer drug, was used to
investigate the effect of the synthesized nanoparticles’ character-
istics on the drug encapsulation and the in vitro release profile. To
encapsulate the drug, PTX (0.1 mg.mL1) was mixed with the HMCs
solutions (1 mg mL1) and injected into the microfluidic device as
the core flow. The acidic media has been shown to prevent PTX
from degradation [37]. Loading efficiency was determined as the
amount of loaded drug in the nanoparticles over the initial amount
of PTX in percent.
To determine the in vitro drug release patterns, PTX-loaded
nanoparticles (1 mg) were dispersed in 1 mL phosphate buffered
saline (PBS, pH 7.4), and injected into a dialysis cassette (3.5K
MWCO, Thermo Scientific). The cassette was then immersed in PBS,
gently shaken and incubated at a temperature of 37  C. At pre-
determined time intervals, samples were collected and measured
with high-performance liquid chromatography (HPLC). In this
experiment, 1 mL sample was mixed with 1 mL acetonitrile; a
reverse phase C18 column was used as the stationary phase, and the
mobile phase consisted of a mixture of acetonitrile and: water
(60:40 v/v). Separation was performed at a flow rate of 1 mL min1,
and PTX was detected at a wavelength of 230 nm.
3. Results and discussion
3.1. Synthesis of nanoparticles by hydrodynamic flow focusing
A cross-junction microfluidic channel was designed to create a
hydrodynamic flow focusing device. As schematically shown in
Fig. 1a, the microchip consists of three inlets (two for basic water
and one for the HMCs solution) and one outlet for the self-
assembled nanoparticles. To provide stable mixing through inter-
facial diffusion between the sheath and the core flows in the
microfluidic devices is of prominent importance in this approach
[21,30]. For this purpose, a rhodamine B solution was squeezed
between the streams of fluorescein sodium, and rhodamine B was
detected with fluorescence microscopy to determine the range of
stable flow rates, which were adjusted with micropumps in the
hydrodynamic focusing region (Fig. 1b).
The synthesized HMCs derivatives in acidic media can be
considered as hydrophobic polyelectrolytes, comprising of alter-
nating protonated amine groups (eNH3þ ) and grafted hydrophobic
side chains. Taking into account that the pKa for chitosan is ca. 6.3e
6.4 [24], the balance between electrostatic repulsion and the hy-
(2)
drophobic attraction forces is drastically influenced by pH. As pH is
increased through diffusion-mediated mass transfer into the
focused polymer stream, HMCs chains are deprotonated and
therefore the hydrophobic interactions as well as possible
hydrogen bonds among the hydroxyl and the uncharged amine
 E. Dashtimoghadam et al. / Polymer 54 (2013) 4972e4979
groups grow in importance and cause the chains to form self-
assembled structures [34]. In fact, as the contacts between the
hydrophobic moieties and water molecules are energetically un-
favorable, the HMCs chains have a strong inter- and intramolecular
tendency to self-assemble into complexes; this process occurs onto
the microfluidic platform by increasing pH to the physiological pH
in a controlled manner.
3.2. Hydrodynamic size and morphology of the nanoparticles
Fig. 2 shows the change of the hydrodynamic diameter of the
self-assembled nanoparticles, formed from HMCs chains with
different hydrophobicity at various flow ratios. As can be seen, both
the degree of hydrophobicity of chitosan and mixing time affect the
size of the nanoparticles. In other words, the size of the nano-
particles can be modulated on-chip by adjusting the mixing time
employed in the particle preparation. The diameter of the nano-
particles increases with increasing flow ratio, while at the same
flow ratio the size decreases with enhanced hydrophobic substi-
tution of pendant hydrophobic groups. This observation is consis-
tent with reported results [29,30], and this finding is attributed to
the stronger hydrophobic interaction between the conjugated C12
groups with increasing hydrophobicity. Although this interpreta-
tion seems rational, it has only been addressed qualitatively [29]. To
further elucidate this phenomenon, it will be discussed on a more
quantitatively basis below, regarding the particle compactness and
chain aggregation number.
Over the whole flow ratio range and degrees of hydrophobic
modification of chitosan, the on-chip generated particles were
found to be smaller than the corresponding nanoparticles prepared
by the conventional bulk mixing method. This implies that there is
a time scale associated with the pH-triggered rearrangement and
self-assembling of the HMCs chains during the growth of the
nanoparticles, which is intimately affected by the controlled flow
ratio on the microfluidic platform. Moreover, the values of the
polydispersity index (PDI) for the microfluidic nanoparticles were
observed to be much lower (PDI < 0.16) than those determined
(PDI > 0.45) from the bulk synthesis. The broader particle size
distribution from the bulk method is due to the residence time
distributions caused by turbulence and convective mixing [12]. On
the other hand, the reproducible synthesis of narrow size distrib-
uted HMCs nanoparticles in the case of hydrodynamic flow
Fig. 2. Effects of flow ratio and time of mixing on the hydrodynamic diameter of
microfluidic synthesized HMCs nanoparticles (Mean  SD, n ¼ 5 independent
experiments).
4975
focusing is ascribed to the microfluidic imparted controllable self-
assembly process. Under microfluidic mixing, deprotonation of
amine groups on HMCs chains takes place even before the poly-
mers commence to form aggregates. This suggests that the nano-
particle assembling process proceeds in a medium that closely
match the final pH, i.e., the physiological condition (pH ¼ 7.4) in
this study. A similar argument was utilized in the case of a
microfluidic-directed nanoprecipitation process [20]. In the course
of bulk mixing, the time scale of nanoparticle assembling is smaller
than the time for a pH change. Thus, nanoparticle assembling oc-
curs in a suspension at non-equilibrium conditions, where the final
pH value has not been reached and augmented structural hetero-
geneity is promoted. To obtain a narrow particle-size distribution
requires rapid nucleation followed by growth of nanoparticles in
the absence of further nucleation, a process that can be accom-
plished by microfluidic flow focusing.
To examine the size and morphology of the synthesized HMCs
nanoparticles, they were examined via atomic force microscopy
(Fig. 1c) as well as transmission electron microscopy (TEM)
methods. A comparison of the TEM illustrations in Fig. 3c and
d clearly discloses spherical shape with a narrow size distribution
for microfluidic synthesized nanoparticles, whereas the corre-
sponding bulk synthesized particles are larger with a visible devi-
ation from the spherical shape and with a broader size distribution.
Based on the analysis of the TEM images, the diameters of the
nanoparticles prepared at a flow ratio of 0.031 were found to be
119  4, 79  3, and 73  4 nm for HMCs-1, HMCs-2 and HMCs-3,
respectively, and 216  12, 208  13, 169  12 for the corresponding
nanoparticles prepared by the bulk mixing method. The obtained
sizes are in good agreement with those determined from the dy-
namic light scattering measurements (cf. Fig. 2).
3.3. Compactness of the nanoparticles
It can generally be argued that the hydrodynamic size of the
colloidal polymeric nanoparticles is governed by the polymer
contents as well as their swelling state. As indicated above, the size
of the HMCs nanoparticles at various mixing times is controlled by
the intricate competition between intra- and intermolecular in-
teractions. This makes the interpretation of particle size changes
complex, because the size can be affected by either swelling or the
aggregation number of chains. Hence, to gain an insight into the
effect of mixing time and hydrophobicity on the characteristics of
HMCs nanoparticles, it is necessary to address their compactness
through the local polymer concentration inside the nanoparticles.
Recently a method, based on the Mie theory for estimating the
local polymer concentration inside spherical nanoparticles, was
developed [32,38]. In the framework of this approach, valuable
information about the self-assembled HMCs nanoparticles can be
extracted by knowing the size of the nanoparticles and the
turbidity of their suspension.
Equation (3) below is employed to calculate the local polymer
concentration, cNP, inside the synthesized HMCs nanoparticles [38],

 
3ct 2 1
s¼ 1 sinðwcNP Þ  ½1  cosðwcNP Þ
2cNP Rh wcNP wcNP
(3)
where s, ct, Rh are the turbidity of the nanoparticle suspension, the
total polymer concentration in suspension, the hydrodynamic
radius of the particles, respectively, and w ¼ 4pRh(dn/dc)/ln0
wherein l is the wavelength for the turbidity measurements, n0 is
the refractive index of the solvent, and dn/dc is the refractive index
increment of the polymer. Since this method has been elaborated
for spherical particles with a narrow size distribution, low PDI
4976 E. Dashtimoghadam et al. / Polymer 54 (2013) 4972e4979

Fig. 3. TEM images of microfluidic synthesized nanoparticles based on HMCs-1 (a), HMCs-2 (b), and HMCs-3 (c) at a flow ratio of 0.031 and HMCs-3 nanoparticles prepared via the
bulk mixing method (d); (scale bar 100 nm).

of the microfluidic synthesized HMCs nanoparticles fulfill this
prerequisite.
The calculated cNP values from Eq. (3) for the synthesized
nanoparticles composed of HMCs chains with different hydropho-
bicity at various flow ratios are displayed in Fig. 4. The results reveal
that at a given flow ratio, more compact nanoparticles are evolved
with increasing hydrophobicity of the HMCs chains. The enhanced
80
0.3
Time of Mixing (ms)
local polymer concentration inside the nanoparticles composed of
more hydrophobic chains, can be attributed to the higher sticking
probability of hydrophobic segments to form intermolecular asso-
ciation structures at the same time scale. However, the difference in
hydrophobicity of the samples seems to play a less role at higher
values of the flow ratio. We also note that the compactness of the
nanoparticles drops with increasing flow ratio; this trend is less
pronounced as the hydrophobicity of the sample is reduced. This
finding may be interpreted in terms of the competition between
intra- and intermolecular interactions at various mixing times. As
shown in the inset of Fig. 4, smix rises with increasing flow ratio; this

60
trend favors the probability of hydrophobic side chains to form
40
intramolecular associations, especially when the hydrophobicity of
the molecules increases and this leads to the formation of less
20
compact particles. A similar effect was observed in the case of a
CNP (g/ml)

0.2
0 reported [39] viscosity drop of semidilute amphiphilic chitosan
0.025 0.050 0.075 0.100 0.200 0.225 solutions. In the cited work, the effect was ascribed to the formation
Flow Ratio
of intramolecular loops [39]. However, it should be noticed that due
to the lower polymer concentrations and rapid mixing times in this
0.1 study, HMCs chains are prone to form intramolecular interactions
even at lower contents of hydrophobic groups. Despite consider-
HMCs-1 ably higher PDI for the bulk synthesized particles, their compact-
HMCs-2 ness was roughly estimated by means of Eq. (3). The lower
0.0 HMCs-3 compactness values for the bulk synthesized particles as compared
to the microfluidic nanoparticles, confirm the inherent problem
0.025 0.050 0.075 0.100 0.200 with the bulk synthetic method to produce dense nanoparticles. It
should be noted that even at the highest considered flow ratio of
Flow Ratio 0.2, the microfluidic synthesized particles are more compact than
Fig. 4. Local polymer concentration (cNP) inside the HMCs nanoparticles as a function
the corresponding bulk prepared nanoparticles. It is worth to notice
of the flow ratio for the systems indicated. The inset shows the change of mixing time that the microfluidic synthesized nanoparticles in this work are
vs. flow ratio. The lines are only guides for the eyes. found to be more compact than the chitosan nanoparticles formed
 E. Dashtimoghadam et al. / Polymer 54 (2013) 4972e4979 4977

in the presence of sodium tripolyphosphate (STPP) [31]. These re-

2.5x10
sults corroborates with the hypothesis that the hydrodynamic flow 7x10
2 HMCs-1
HMCs-2 HMCs-1
HMCs-3
focusing approach employed in this work produces particularly 2.0x10
HMCs-2
compact nanoparticles, especially for chitosan of high degree of 2 HMCs-3
6x10
hydrophobic modification and at low flow ratios. The overall pic- 1.5x10

N
ture that emerges from Fig. 4 suggests that the particle compact- 1.0x10
2
ness can be tuned by altering the hydrophobicity of the chitosan 5x10
sample and the flow ratio. By modulating these parameters, 5.0x10
0.025 0.050 0.075 0.100 0.200 0.225
nanoparticles for various applications can be designed. 4x10
2
Flow Ratio

Nagg
2
3.4. Aggregation number of HMCs chains in the nanoparticles 3x10

2
The results in Figs. 2 and 4 show that the size of the nano- 2x10
particles is increased while their compactness decreases with
(a)
increasing mixing time. This can be due to swelling and/or an 2
1x10
increase of the number of aggregated chains inside the HMCs 0.025 0.050 0.075 0.100 0.200
nanoparticles. To discriminate between these two coexisting phe-
nomena, the aggregation number of polymer chains is examined Flow Ratio
here to give some insights into the role of mixing time on the self-
assembly of HMCs chains. 13
2.5x10 HMCs-1
From the determination of cNP with the aid of Eq. (3), we can
HMCs-2
calculate the molecular weight of the spherical particles, MNP, form
HMCs-3
the following expression,
13
4 3 2.0x10
MNP ¼ pR c N (4)
N (particles/ml)
3 h NP A

where NA is Avogadro’s number. The aggregation number of poly-

mer chains in the nanoparticles, Nagg, can be determined from 1.5x10
13

Eq. (5),

MNP
Nagg ¼ (5)
Mn
where Mn is the number average molecular weight of the polymer.
The aggregation of HMCs chains into nanoparticles is generally
governed by the local inter- and intramolecular interactions be-
tween the hydrophobic attracting sites, which constitutes of hy-
drophobic side chains and uncharged acetylated units inherent to
chitosan itself. Upon microfluidic mixing, rapid nucleation of ag-
gregates is triggered through deprotonation of HMCs chains,
leading to the formation of nanoparticles. Fig. 5a shows that the
aggregation number rises as the flow ratio is increased and it flat-
tens out at high flow ratios. Actually, the number of aggregated
chains becomes independent of flow ratio with increasing mixing
time, and this occurs at lower flow ratios in the case HMCs-3. The
conjecture is that the intrapolymer association intensity of the
HMCs chains is strengthened with increasing hydrophobicity of the
polymer. Since this effect is more prominent for HMCs-3, interchain
association of the polymer chains is repressed. The results suggest
that at lower flow ratios mixing occurs faster than the time scale for
aggregation, i.e., smix < sagg, and this leads to a lower number of
kinetically locked HMCs chains inside the nanoparticles.
Simple calculations disclosed that despite quite different num-
ber of chains inside the aggregates, the number of grafted hydro-
phobic stickers in each aggregate, NStickers, is roughly the same for
the HMCs-2 and HMCs-3 samples at flow ratios higher than 0.05
(see the inset of Fig. 5a). This means that the polymer chains in a
complex associate in such a way that independently of the number
of chains, each aggregate contains virtually a constant number of
associating groups to acquire a necessary gain in the energy for
association [40]. The association energy is determined by the
number of attracting hydrophobic groups. In view of this, HMCs
chains with a smaller number of associating groups should aggre-
gate more to provide the sufficient number of associating groups to
13
1.0x10
(b)
0.025 0.050 0.075 0.100 0.200
Flow Ratio
Fig. 5. Aggregation number (Nagg) of HMCs chains in the nanoparticles as a function of
flow ratio for the systems indicated. The inset shows the number of hydrophobic
stickers (NStickers) inside the corresponding HMCs nanoparticles (a). The number
density (N) of HMCs nanoparticles synthesized at various flow ratios (b). The lines are
just guides for the eyes.
stabilize the aggregate. This result is consistent with the results
reported previously for the multichain aggregates of associating
polyelectrolytes [28].
By comparing the calculated number of aggregated chains with
the size and compactness of the nanoparticles, it can be inferred
that two mechanisms are decisive for the size and compactness of
the particles, namely unimer addition and chain rearrangement
due to the interplay between intra- and intermolecular in-
teractions. Indeed, an increase of the hydrodynamic radius while
the compactness of the HMCs nanoparticles is decreased can
initially be controlled by raising the number of aggregated chains,
and gradually the arrangement of the self-assembled chains turns
out to have an impact on the process. In this regard, tunable
microfluidic mixing has enabled systematically varying flow rates
in order to optimize the aggregation and self-assembly of the HMCs
chains.
Another characteristic feature of the nanoparticulate systems is
the number density of the particles (number of particles per unit
volume of the suspension). However, despite its importance from a
practical point of view, number density values of nanoparticles
are seldom reported due to experimental difficulties. From the
4978 E. Dashtimoghadam et al. / Polymer 54 (2013) 4972e4979

determined compactness of the nanoparticles, the number density 100

of the particles, N, can be estimated by using Eq. (6), HMCs-1
HMCs-2
3ct
N ¼ (6) 80 HMCs-3
4cNP pR3h

Loading Efficiency (%)

Fig. 5b shows that the number density of HMCs particles N (this
quantity is directly related to the corresponding aggregation 60
number) increases with increasing hydrophobicity of the chains.
Polymeric nanoparticles are of great interest for controlled drug
delivery systems, and one of the factors that have a profound effect 40
on the release profile of the loaded drugs is the degree of swelling
of the particles. It has been observed that swollen nanoparticles
with open structure exhibit increased release rate [6,31]. In view
20
of the size of the synthesized HMCs nanoparticles, they can be
considered as potential passive targeting drug delivery vehicles, Bulk
providing enhanced permeability and retention effect to be accu- (a) Mixing
mulated at the tumor sites [2,4,5]. To explore the effect of the 0
microfluidic potential for loading as well as release behavior of 0.00 0.05 0.10 0.2
drugs, the encapsulation efficiency and diffusion coefficient of Flow Ratio
paclitaxel (PTX) loaded in HMCs nanoparticles were investigated
(Fig. 6).
Encapsulation efficiency is defined as the fraction of initial PTX 100 (b)
that is encapsulated by the nanoparticles. It is shown in Fig. 6a that
at the same flow ratio, encapsulation is increased with increasing
hydrophobicity of the HMCs nanoparticles, whereas encapsulation 80
Cumulative Release (%)

decreases with increasing mixing time. As expected, the loading

capacity of the drug is correlated to the substitution degree of the
hydrophobic groups; higher hydrophobicity of the particles pro- 60 40 60

Diffusion coefficient×10 (m s )
97nm
motes higher loading capacity of the hydrophobic drug. Moreover,
30
it is found that more drug molecules are entrapped inside the 40
nanoparticles at lower flow ratios, which may be attributed to the 40

t50 (h)
20
fact that more hydrophobic moieties are exposed to the drug
molecules during the shorter mixing times to interact with the PTX F.R. 0.031 61nm
20

molecules in the course of nanoparticles formation. 20 10

F.R. 0.075 37nm 48nm
The in vitro release from the synthesized drug-loaded HMCs-3 F.R. 0.2 0 0
Bulk
nanoparticles (chosen because of its higher loading capacity) at 0.00 0.05 0.10 0.2 Mixing 0.3
0 Bulk Mixing
flow ratios of 0.03, 0.075 and 0.2, were compared with the corre- Flow Ratio

sponding bulk-synthesized nanoparticles to establish the effect of
using rapid mixing to tune the release profile and the diffusion
behavior of drug molecules. Fig. 6b shows the release profiles of
PTX over two weeks. The release behavior demonstrates a relatively
high release rate at the initial stage, followed by an asymptotic
release pattern. The release in the initial stage may be connected
with the drugs loaded in the corona of the nanoparticles [30]. It is
obvious that the microfluidic nanoparticles exhibit a slower release
rate compared to the bulk synthesized analogs. One may also
analyze the release kinetics in terms of the fraction of released drug
and the square root of time according to Eq. (7) [41],
!1=2
Mt Dt
¼ 6
MN pR2h
0 50 100 150 200 250 300
Time (h)
Fig. 6. Drug loading efficiency of microfluidic synthesized HMCs nanoparticles con-
taining paclitaxel (PTX) in comparison with nanoparticles prepared from the bulk
mixing method (a). Release profiles of PTX from HMCs-3 nanoparticles at flow ratios of
0.031, 0.075, and 0.2 compared with the corresponding bulk synthesized nanoparticles.
The inset shows estimated values for the diffusion coefficient, the time of 50% drug
release (t50), and measured hydrodynamic radius of the HMCS-3 nanoparticles at
various flow ratios (b) (Mean  SD, n ¼ 5 independent experiments).
stickers participating in synthesis of the corresponding nano-
particles (the inset of Fig. 5a). These results indicate the crucial role
of the time-frame of self-assembling in controlling loading and the
(7)
release behavior of the resultant nanoparticles.

where Mt/MN is the fraction of released drug at time t, D is the

diffusion coefficient of the drug molecules and Rh is the hydrody-
namic radius of the particles. The estimated values for the diffusion
coefficient, the time of 50% drug release (t50), and the measured
hydrodynamic radius of the HMCS-3 nanoparticles are all displayed
in the inset of Fig. 6b. It is evident that microfluidic nanoparticles
show considerably lower values of the diffusion coefficient along
with extended t50. These results are compatible with the more
compressed nanoparticles formed in the rapid mixing regime.
Although the value of the diffusion coefficient was found to rise
with increasing mixing time, the relatively longer t50 at a flow ratio
of 0.075 may be ascribed to the higher number of hydrophobic
4. Conclusions
The self-assembly of amphiphilic polymers into colloidal
nanostructures has inspired research in many aspects of materials
science. The combination of the amphiphilic nature of such self-
organized structures with the possibility of in situ incorporation
of active ingredients have enabled them to be served as promising
nanocarriers for biomedical applications. To proceed toward
developing nanoparticles (NPs) with customized and reproducible
properties, we explored the possibility of rapid and tunable mixing
to control the characteristics of self-assembled NPs, based on
 E. Dashtimoghadam et al. / Polymer 54 (2013) 4972e4979 4979

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