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Colloids and Surfaces B: Biointerfaces 82 (2011) 81–86

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

 j o u r n a l h o m e p a g e :   w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f b

Smart swelling biopolymer microparticles by a microfluidic approach:

Synthesis, in situ encapsulation and controlled release

Aiping Fang∗, Bernard Cathala

BIA-NANO, INRA, Rue de la Géraudière, BP 71627, 44316 Nantes, France

a r t i c l e i n f o

 Article history:

Received 9 July 2010

Received in revised form 12 August 2010Accepted 13 August 2010

Available online 21 August 2010

Keywords:

Microfluidic synthesis

Biopolymer microparticles

Rapid mixing

In situ encapsulation

Smart swelling

Controlled release

a b s t r a c t

This paper reports a microfluidic synthesis of biopolymer microparticles aiming at smart swelling.

Monodisperse aqueous emulsion droplets comprising biopolymer and its cross-linking agent were

formedin mineral oil and solidifiedin thewinding microfluidic channelsby in situ chaotic mixing, which

resulted in internal chemical gelation for hydrogels. The achievement of pectin microparticles from  in

situ mixing pectin with its cross-linking agent, calcium ions, successfully demonstrates the reliability of 

this microfluidic synthesis approach. In order to achieve hydrogels with smart swelling, the following

parametersand theirimpactson the swellingbehaviour, stability andmorphology of microparticles were

investigated: (1) the type of biopolymers (alginate or mixture of alginate and carboxymethylcellulose,

A–CMC); (2) rapid mixing; (3) concentration and type of cross-linking agent. Superabsorbent micropar-

ticleswere obtained from A–CMC mixture by using ferric chloride as an additional external cross-linking

agent. The in situ encapsulation of a model protein, bovineserum albumin (BSA), was also carried out. As

a potential protein drug-delivery system, the BSA release behaviours of the biopolymer particles were

studied in simulated gastric and intestinal fluids. Compared with alginate and A–CMC microparticles

cross-linked with calcium ions, A–CMC microparticles cross-linked with both calcium and ferric ions

demonstrate a significantly delayed release. The controllable release profile, the facile encapsulation as

well as their biocompatibility, biodegradability, mucoadhesiveness render this microfluidic approach

promising in achieving biopolymer microparticles as protein drug carrier for site-specific release.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Since 1990s, with the emerging technology in miniaturisa-

tion and MEMS, microfluidics has been considerably diversified

for widespread applications in the multidisciplinary fields of 

chemistry, biology and physics   [1].   Microfabrication by silicon

technologies  [2]   and the recent soft lithography   [3]  has greatly

promoteda microfluidic routeto tackle the questionsin bothfunda-

mentals and applications due to its low cost, low power or reagent

consumption and high performance. Specifically, the new develop-

ments in microfabrication techniques have enabled the fabrication

of very efficient emulsification microstructured devices that allowemulsifying a fluid in another immiscible fluid [4]. Thus, droplets

[5] or bubbles [6,7] can be continuously produced and dispersed

in a continuous fluid flowing within these microfluidic devices.

If the as-generated droplets or bubbles can be solidified down-

stream either thermally or chemically, one achieves synthesis of 

microparticles by a microfluidic route. In fact, continuous efforts

have been dedicated to this approach since the first demonstra-

∗ Corresponding author. Tel.: +33 240675068; fax: +33 240675043.

E-mail addresses: aiping.fang@nantes.inra.fr, aiping.fang@gmail.com (A. Fang).

tion of the controlled formation of micrometer-sized oil-in-water

(O/W) and water-in-oil (W/O) emulsion droplets in a microma-

chined silicon device [8]. The high potentialities of the microfluidic

fabrication approach stem from the possibility to generate highly

monodisperse droplets (the coefficient of variation of the particle

size distribution, c.v., is typically lower than 5%), one at a time with

an incomparable degree of control over size. Additionally, in the

droplet formation, the ability to control the local flow field via fab-

rication of complex microscale geometriesenables control over the

deformation and breakup of everyindividual droplet, thus allowing

control over the shape, morphology, internal structures [9], chem-

istry (isotropic and anisotropic/Janus particles)  [10–12].  It is theonly technique which enables a 100% encapsulation and a control

over the nature of encapsulated objects in a single step  [13,14].

Polysaccharides, such as chitosan, alginate, pectin, cellulose,

are naturally occurring carbohydrate-based biopolymers. They are

non-toxic and offer high water solubility, biocompatibility and

biodegradability  [15].   One feature of these biopolymers is their

high content of functional groups (e.g. amino groups in chitosan;

carboxylic groups in pectin and alginate). These functional groups

can be utilised for cross-linking, resulting in fabrication of func-

tional microgels of biopolymers. Pectin and alginate, for example,

areknownto form complexeswith divalent ions, such as Ca2+, Ba2+,

0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.colsurfb.2010.08.020

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82   A. Fang, B. Cathala / Colloids and Surfaces B: Biointerfaces 82 (2011) 81–86

andSr2+ in aqueous solution.In addition,those polymerswith func-

tional groups such as amino groups and carboxylic acid groups are

pH-sensitive [16–18].  The synthesis of microparticles from those

biopolymers has long been the focus of a very intensive research in

biomedicine, pharmaceutics, material science, cosmetics and food

industry [19,20].

On one hand, microfluidics has recently emerged as a very

promising route to the controlled synthesis of polymeric particles.

On the other hand, few works have been done by a microflu-

idic route in the research field of stimuli-responsive biopolymer

microparticles toward controlled release. In this work, we develop

a microfluidic route to synthesize and fabricate stimuli-responsive

microparticles from naturally occurring polysaccharides. Microflu-

idic devices from poly(dimethylsiloxane) (PDMS) with versatile

control over the local fluids and their mixing in microchannels by

complex microscale geometries were developed for the fabrication

of polysaccharide microparticles. The emulsified polysaccharide

droplets consisting of cross-linking agent were in situ solidified by

rapid mixing while traveling in winding microfluidic channels.Due

to the versatile control over the local fluids, bi-polymer micropar-

ticles including alginate and carboxymethylcellulose (CMC), or

bi-polymer microparticles encapsulated with a model drug, bovine

serum albumin (BSA), were able to obtain in situ with the microflu-

idic devices. Their smart swelling and release behaviours indifferent pH conditions were examined and discussed. Together

with the ability to control over chemical anisotropy, shape com-

plexity and size by microfluidic techniques, this microfluidic

approach will shed new light in the research for the biocom-

patible and biodegradable microparticles with stimuli-responsive

behaviour, which we believe will find wide applications in cosmet-

ics, pharmaceutics and tissue engineering.

2. Experimental

 2.1. Materials

Pectin (P, deacetylation ∼70%, from citrus fruit), ferric chloride

(reagent grade), calcium chloride (reagent grade), BSA, surfac-tants Tween 20 and Span 80 were purchased from Sigma–Aldrich.

Alginate (A) with molar mass of 94,400 g/mol and sodium car-

boxymethylcellulose (CMC) with molar mass of 31,000g/mol were

generous gifts from Cargill and Aqualon, respectively. BSA conju-

gated with fluorescent dye FITC (BSA-FITC) was synthesized and

purified by dialysis in our lab. Filtered water from a Milli-Q sys-

tem was used for the preparation of all aqueous solutions. Mineral

oil with 2% Span 80 (both from Sigma) was used as continuous oil

phase to produce droplet-based emulsion in microfluidic devices.

 2.2. Microfluidic fabrication of microparticles

 2.2.1. Fabrication of PDMS microfluidic devices

Procedures for the fabrication of PDMS structures for microflu-

idics have been described in detail elsewhere  [21].   Briefly, the

design of the microstructures was made in a Adobe Illustrator

(AI) software and printed out on transparencies as photomask in

UV-lithography, resulting in bas-relief structure from a photore-

sist SU-8 on silicon wafer which serves as a master for fabricating

PDMS molds. To create the PDMS mold, a liquid PDMS prepoly-

mer (in a mixture of 1:10 base polymer:curing agent) was poured

onto it. The PDMS was cured at 70 ◦C for 1 h or more and peeled

off the master, producing the final replica bearing the designed

microstructures. Small holes were drilled into the PDMS using a

borerto produce fluidinlets and outlets.Finally,PDMS devices were

formed by irreversibly bonding the PDMS replica with a flat PDMS

slab (in a mixture of 1:20 base polymer:curing agent) at 70 ◦C for

2 h or more.

 2.2.2. Microfluidic fabrication of biopolymer particles

Biopolymer microparticles were achieved by emulsifying aque-

ous solution of biopolymer and its cross-linking agent in mineral

oil as the continuous oil phase in the microfluidic devices as

shown in Fig. 1. The two immiscible liquids were supplied to the

microchannels using digitally controlled syringe pumps (Harvard

Apparatus PHD 2000, USA). The continuous oil phase was deliv-

ered to inlet 1 (I1). The water phase flow was achieved by in situ

mixing of three aqueous streams: a biopolymer solution (I2), its

cross-linking agent solution (I4) and inert water (I3). Typically,

to achieve pectin or alginate microparticles, inlet 2 (I2), inlet 3

(I3) and inlet 4 (I4) were supplied with 5 wt% pectin or 4 wt%

alginate solution, water and 1.0wt% calcium chloride solution,

respectively. The aqueous liquid phase broke up in continuous oil

phase to generate W/O emulsion droplets. As a rule of thumb,

the concentration of cross-linking agent was chosen in such a

way that it should not be too high to generate instant gelation

at breakup cross-junction. The size of the droplets can be con-

trolled by varying the flow rate of the two immiscible liquids.Cross-linking was induced by rapid mixing of the ingredients

inside the droplets while they travel in the winding microchannels

downstream. To achieve a bi-biopolymer alginate–CMC (A–CMC)

particles, instead two biopolymer, alginate and CMC, were deliv-

ered to I2 and I4. The microparticles were collected from the

microfluidic devices with a large volumebuffer solution under gen-

tle agitation.The detailedexperimental conditionsfor the synthesis

of each biopolymer microbead sample and its coding informa-

tion are listed in Table 1. Specifically, external cross-linking with

Fig. 1.   Optical microscope image of microfluidic emulsification and schematic of microfluidic devices used in this work. (a) Immiscible liquids are delivered to microfluidic

channels by inlet 1 (I1, continuous oil phase) and inlets 2–4 (I2, I3, I4, disperse aqueous phase). The biopolymer microparticles are collected from outlet 5 (O5). Winding

microchannels(b) without and (c) withadditional inlet6 (I6)is designedfor dilution the droplet trainwith continuous phase. The microchannelshave a square cross-section

with 100m by width and 78m by height. The optical microscope image is the zoom-in area as indicated by the dotted square in (a).

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 Table 1

Experimental conditions for synthesis and encapsulation of microparticles on

microfluidic devices.

Sample code Aqueous phase Collecting

medium

I2 I3 I4

P 5% P Water 1.0% CaCl2   1a

A 4% A Water 1.0% CaCl2   1

A–CMC 4% A Water 1.0% CaCl2,

0.5% CMC

1

A/CMC–Fe 0.8% A, 4% CMC Water 1.0% CaCl2   2b

A–CMC–Fe 4% A Water 1.0% CaCl2,

0.5% CMC

2

A–BSA 4% A 1.0% BSA 0.5% CaCl2   1

A–CMC–BSA 4% A 1.0% BSA 0.5% CaCl2,

0.5% CMC

1

A/CMC–Fe–BSA 0.8% A, 4% CMC 1.0% BSA 0.5% CaCl2   2

A–CMC–Fe–BSA 4% A 1.0% BSA 0.5% CaCl2,

0.5% CMC

2

a Microparticles werecollected witha solutionof 0.050 M acetatebufferwith 10%

CaCl2, 3% Tween 20 (pH 5.0).b Microparticles were collected with a solution of 2% FeCl 3   in 0.050 M acetate

buffer with 10% CaCl2, 3% Tween 20 (pH 5.0).

ferric ions was achieved by collecting the microparticles in thepresence of 2% ferric chloride in the buffer solution. Micropar-

ticles were extracted and washed with 0.050M acetate buffer

with 3 vol.% Tween 20 (pH 5.0) by centrifuging the dispersion

at 2000 g   for 2 min. For release studies or swelling studies, they

were further transferred to different simulated physiological fluids

(SPFs).

 2.3. Swelling studies of microparticles

An Olympus IX51 inverse microscope (Olympus) equipped with

a digital camera (Sony, SCD-SX90) was used to acquire images.

The swelling behaviour was studied by measuring and compar-

ing the diameters of microparticles when they reached swelling

equilibrium. To study the effect of pH on swelling, particles were

dispersed under gentle agitation in 0.1 M HCl/NaCl buffer solution,

pH 1.2, as simulated gastric fluid (SGF), and in 0.050M potas-

sium acetate/acetic acid, pH 5.0, as simulated gastrointestinal fluid

(SGIF), and in 0.050M KH2PO4/NaOH, pH 7.4, as simulated intesti-

nal fluid (SIF)   [22].   The swelling ratio (SR) has been calculated

before as SR%=100 (Ds −Dd)/Dd, where the   Ds   and   Dd   are the

diameters of dry and swelled particles, respectively   [23].   In our

case, however, thedriedmicroparticles areoften notsphere, which

makes it difficult to measure their size. As a result, we calculated

their relative swelling ratio (RS) as following: RS5.0/1.2 = D5.0/D1.2,

where RS5.0/1.2 denotes the swelling of beads at pH 5.0 relative to

that at pH 1.2; D5.0 and  D1.2 are their diameters at pH 5.0 and 1.2,

respectively. Accordingly, RS7.4/1.2  indicates the swelling ratio of 

microbeads at pH 7.4–1.2.

 2.4. In vitro BSA release studies

The freshly prepared microparticles encapsulated with BSA

were dispersed in 5.0 mL of 0.050M KH2PO4/NaOH solution (pH

7.4) at 37 ◦C using a shaking water bath (50 rpm). 1.0mL of sam-

ple solution at appropriate intervals was withdrawn and replaced

each time by same amount of fresh buffer solution. The amount of 

BSA in the release medium was analysed by UV spectrophotome-

try (SPECORD S600, AnalytikJean) at 280nm. The release assay was

reproduced 3 times to obtain the average value. The unchangeable

amount of BSA released in the solution after 48 h can be presumed

as the preloading amount of BSA in microparticles [24]. The cumu-

lative protein release was calculated as following:

Cumulative release (%) =Amount of released BSA

Amount of preloaded BSA  × 100

The BSA release profiles were plotted as the cumulative amounts

in the release medium against the release time.

3. Results and discussion

 3.1. Microfluidic synthesis and in situ encapsulation

Due to its ionotropic gelation with Ca2+ and its wide range of 

applications, pectin was chosen as a model biopolymer to demon-

strate the reliability of this microfluidic synthesis approach.  Fig. 1

shows the image of microfluidic emulsification when pectin was

used as a model biopolymer. When pectin and divalent Ca2+

solutions are mixed in the emulsion droplet, the gelation and

cross-linking of the polymers are achieved by the exchange of 

sodium ions from galacturonic acids with calcium ions to form

stable 3-D structures.   Fig. 2a   depicts the monodisperse pectin

beads with a diameter of 100m fabricated through the microflu-

idic device shown in   Fig. 1a. The introduction of a jet of inert

water flow sandwiched between pectin and its cross-linking agentsolution helps to avoid instant gelation at the cross-junction

which may cause unsmooth breakup or even clogging. The chem-

ical gelation was controlled by the flow rate ratio of the three

aqueous streams. Along with varying the flow rate of continu-

ous phase, droplet size can be controlled   [25].   The size of the

Fig. 2.   Optical microscope images of pectin microparticlesfabricated from(a) strait

and (b) winding microchannels as shown in  Fig. 1a and c, respectively. Flow rates

used in (a) are I1= 1.50 mL/h, I2 =I4 = 0.05 mL/h, I3= 0.08mL/h, those in (b) are

I1= I6= 2.0 mL/h, I2= I4= 0.03mL/h, I3=0.07mL/h. Scale bar: 50m.

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84   A. Fang, B. Cathala / Colloids and Surfaces B: Biointerfaces 82 (2011) 81–86

microparticles usually falls in the range of 40–100m from our

experiments.

Pectin beads with high monodispersity (c.v.= 4.5%) were repro-

ducibly obtained with a device shown in   Fig. 1b. Their shape,

however, was not uniform. As observed from Fig. 2a, parts of the

beads are missing, resulting in irregular shape. This is most proba-

bly due to the poor mixing of the pectin with its cross-linker as the

chemical gelation depends greatly on the diffusion of calcium ions

in the biopolymer chain structures. The in situ mixing can be actu-

ally improved by including winding channels due to the fact that

chaotic advection for efficient mixing is generated by the unsteady

fluid flow inside each droplet when it travels through the wind-

ing channels [26]. Results from Fig. 2b confirm this improvement,

where uniform beads were achieved from rapid mixing by apply-

ing the winding microchannels shown in Fig. 1c. Previous work has

demonstrated that, in order to achieve chaotic mixing in winding

channels, the droplet plug should be large enough to “touch” the

walls of the channel [27]. In our experiments, however, we found

that mixing was efficient enough to achieve uniform microparti-

cles as long as the droplets deformed when moving in the winding

channels.

If large microparticles were preferred (for example, diameter

more than 100m),theflow ratesof continuous anddisperse phase

were adjusted accordingly to achieve large droplets. Droplet fusionin large droplet train was often observed, leading to polydispersed

beads. In order to prevent droplet fusion, a stream of continuous

flowwas introduced todilute thedroplettrain from inlet6 asshown

in Fig. 1c [28].

The novel design shown in Fig. 1 including a jet of sandwiched

stream will make encapsulation facile simply by replacing the

water stream with cell or colloid dispersion solution. To achieve

a drug carrier system, a solution of 1.0% BSA as a model pro-

tein drug was supplied to I3 instead of water. Unless otherwise

stated, other conditions remained the same as those to fabri-

cate their BSA-free counterpart as shown in  Table 1.  As droplets

travel downstream, inside each droplet, rapid mixing and chem-

ical gelation take place instantly. The ideal situation is that the

chaotic mixingshould be much fasterthan gelation.When thegela-tion rate is superior to the mixing, droplets are solidified before

efficient mixing is reached, resulting in poorly integrated beads.

The latter can be visually spotted by using BSA-FITC instead of 

BSA as presented in   Fig. 3a. At constant flow rate of both dis-

perse and continuous phases, chaotic mixing rate in the winding

channel is constant.Therefore, this problem canbe solvedby reduc-

ing calcium concentration for a low gelation rate while keeping

chaotic mixing rate constant. A–CMC–Fe–BSA microparticles using

0.5 wt% calcium are presented in   Fig. 3b, showing a totally dif-

ferent structure and morphology from that in Fig. 3a. When the

calcium concentration was further reduced, microparticles with

smoothsurface butvaryingshapewas achieved (imagenot shown),

which could be attributed to the incomplete solidification inside

the microchannels. And incompletely solidified droplets were eas-ily deformed under agitation when they were collected with a

large volume of solution, leading to the polydispersed beads and

poor sphericity. Although the reason is not clear why lower con-

centration of calcium as cross-linking agent had to be used when

encapsulating BSA, the fact that BSA may not be as inert as water

should not be neglected. To achieve uniform and homogeneous

microparticles, 0.5% calcium was applied throughout  in situ   BSA

encapsulation.

 3.2. Smart swelling and controllable release

The pH dependence of the swelling of the microparticles was

investigated by immersing the microparticles in SPFs at room tem-

perature until a swollen equilibrium was reached. Swelling was

Fig. 3.   Confocal fluorescence microscopy images of A–CMC–Fe–BSA hydrogels

obtained byusing(a) 1.0% and(b) 0.5% CaCl2 as cross-linkingagent. All other condi-

tions are listed in Table 1. The zoom-in images of individual beads are also shown.

FITC conjugated BSA was used instead of BSA. Scale bar: 50m.

studied by measuring and comparing the diameters of the swelledmicrobeads. As shown in   Fig. 4,  pectin microparticles have the

same SR at pH 1.2 and 5.0, while alginate particles swell slightly

more at pH 5.0, which is indicated by their RS5.0/1.2 values in Fig. 4.

Neither pectin nor alginate microparticles demonstrate a tunable

smart swelling behaviour. As we are interested in the fabrication

of novel stimuli-responsive functional materials from naturally

occurring biopolymers, efforts were directed toward microparti-

cles with super swelling properties. Inspired by a recent work from

Zhang and coworkers  [29],  we introduced CMC into the alginate

network for bi-polymer microparticles. The bi-polymer particles,

A–CMC, show obviously increased swelling in SPFs (Fig. 4), which

can be attributed to the macropore structures formed by electro-

static repulsion from the highly hydrophilic carboxyl groups in

CMC. Similar to previous work  [29],  it is presumed that alginatecross-linked by Ca2+ acted as a strong backbone in the hydrogel

structure, whereas CMC contributed to the enhanced pore size. As

CMC can be cross-linked by ferric ions [30], a further cross-linking

of CMC in A–CMC hydrogels will alter their swelling behaviour.

When we applied ferric ions as an additional cross-linking agent

forthese bi-polymerbeads,a considerable increase in theirswelling

wasachieved. Their RS5.0/1.2 morethan 1.5 was observed, indicating

superabsorbent behaviours. The swelling of A/CMC–Fe microbeads

at pH 1.2 and 5.0 are also visually compared in Fig. 4.

For all the microparticles, prolonged incubation at SGF (pH

1.2) and SGIF (pH 5.0) did not induce any disintegration of the

microbeads. On the contrary, microparticles except those cross-

linked with ferric ions were almost immediately dissolved in SIF

at pH 7.4. Surprisingly, between microparticles cross-linked with

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Fig. 4. Swelling studies of themicroparticles in differentSPFs. The swellingof A/CMC–Fe beadsat pH1.2 and5.0 arealsovisually comparedin right panels. Scale bar: 50m.

ferric ions but with different methods to mix the two biopolymers,

we found the obvious differences in their swelling behaviour and

stability in SIF. When alginate and CMC were pre-mixed and sup-

plied to microfluidic channels to synthesize A/CMC–Fe microbeads

as detailed in Table 1, they decomposed after an overnight incuba-

tion. When alginate and CMC are supplied to microfluidic channels

through different inlets and mixing was only carried out   in situ

chaotically, the as-formed A–CMC–Fe microbeads show extremestability at pH 7.4. We did not observe any disintegration or

decomposition in the microbeads after an overnight incubation.

The improved swelling by ferric ion cross-linking may stem from

the stable electrostatic interactions between ferric ions and rich

hydrophilic hydroxyl and carboxyl groups in the bi-polymer net-

works of CMC and alginate   [31].  The participation of ferric ions

in cross-linking is demonstrated by the very distinguishable red

color of these microparticles. At this stage, we are not clear why

A–CMC–Fe microbeads are morestable than A/CMC–Fe microbeads

in SIF. It may be closely related to the 3-D structure of the blended

bi-polymer networks. The difference here lies in that, in A–CMC–Fe

microbeads, phase segregation may be dominant; in A/CMC–Fe

microbeads, the CMC and alginate chains remain more intimately

mixed.This differenceis believed tobe directlyresponsiblefor theirdifferent behaviours in hydration [32]. No attempts were made yet

to characterise the microstructures of alginate–CMC bi-polymer

beads in more detail. Itis speculated that phase segregation renders

the bi-polymer beads more stable at pH 7.4.

The relative swelling ratios somehow reflect their swelling

behaviours as the pK a   of the biopolymers used in this work is

larger than 1.2 (the pK a   of alginate and CMC are 4.0 and 3.5,

respectively). Thus, in all these hydrogels at pH 1.2, electrostatic

repulsions between the ionisable groups are minimum, leading to

a minimum uptake of the solvent. As a result, their diameters at pH

1.2 provide a good reference to compare their swelling behaviour.

For those microparticles that are unstable at pH 7.4, their RS5.0/1.2

values reasonably indicate their swelling ability. The maximum

swelling is achieved for A–CMC–Fe microbeads in SIF at pH 7.4,with a RS7.4/1.2 of 1.8 as shown in Fig. 4. Therefore, we define the

swelling capacity as RSm/l = Dm/Dl, where “m” and “l” indicates the

most swollen and least swollen states while the microbead still

maintains its integrated spherical structure. For the A–CMC–Fe

beads, for example, RSm/l is equal to their RS7.4/1.2, while for other

beads, RSm/l is their RS5.0/1.2. As observed in Fig. 4, their swelling

capacity is in the order of P < A < A–CMC< A/CMC–Fe < A–CMC–Fe.

That A–CMC–Fe or A/CMC–Fe beads have the large swelling

capacity in SPFs shows their stability, indicating potential drug-

delivering systems with controlled and sustained release. Those

beads with small swelling capacity, such as P, A, A–CMC, can

be targeted for rapid release. It is worth mentioning that the

bi-polymer beads were formed by a mild cross-linking with-

out involving stringent reactions with hazard reagent, such as

epichlorohydrin. This microfluidic approach provides a reliable

route to the synthesis of biocompatible microparticles with high

control.

Fig. 5   shows the BSA   in vitro   release profiles of the various

test microbeads, namely, A, A–CMC, A/CMC–Fe and A–CMC–Fe

microparticles. Among them, A and A–CMC microbeads show sim-

ilar release behaviour. Significant early release was observed. Their

half release (cumulative release at 50%) was reached in half anhour for A microbeads, in 1 h for A–CMC microbeads. The beads

were fully decomposed after 40h at pH 7.4, leading to full release

of BSA. Contrary to this rapid release, A–CMC–Fe and A/CMC–Fe

microbeads show a different release scenario. Their release was

much delayed, especially for the A–CMC–Fe microbeads, which

reached half release after 30h. The half release of A/CMC–Fe

microbeads wasachieved after5 h,theirfullBSA release byfinaldis-

integration of microbeads was obtained by prolonged incubation

at release medium. However, it was obtained over 3 days. For the

stable A–CMC–Fe microbeads, decomposition of the beads did not

start until 2 days in pH 7.4 release medium, indicated by a change

to red color and abrupt increase of BSA in the release medium.

Their full BSA release, however, was not observed within 1 week.

For the case of A/CMC–Fe microbeads, although the decomposi-tion starts immediately when they were dispersed in the release

medium, the full BSA release was reached in a similar sustained

way as in A–CMC–Fe microbeads. The periods needed for the A,

A–CMC, A/CMC–Fe, A–CMC–Fe microbeads to reach 80% release

vary as followings: 2, 2, 28, 116 h, respectively.

In spite of their different release rates, a noticeable common

phenomenon is that all microbeads demonstrate a rapid burst BSA

release at the beginning release stage, owing to the surface loaded

BSA in hydrogels. After this initial burst period, the BSA release

slowed down andwas dependent on the decomposition of individ-

Fig. 5.   The   in vitro   BSA release profiles from different microparticles in release

medium at 37◦

C. The dotted lines are guide to eyes.

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86   A. Fang, B. Cathala / Colloids and Surfaces B: Biointerfaces 82 (2011) 81–86

ual microbeads. It is found that their release profiles correspond

well to their swelling capacity as discussed earlier. The larger RSm/l

the microbeads demonstrates, the more sustained release was

observed. The results clearly reveal that these microbeads show a

wide rangeof pH-sensitive and release behaviours, thus have appli-

cation potentials in site-specific controlled and sustained release.

4. Conclusions

In this work, we describe a microfluidic approach to pre-

pare novel super swelling hydrogels and  in situ  encapsulation by

versatile control over the local fluids and their rapid chaotic mix-

ing in microchannels. The microbeads achieved by this approach

have a narrow size distribution, good sphericity and controllable

encapsulation. Their sizes ranged from 40 to 100m in diameter

with a c.v. less than 5%. We fabricated alginate–CMC bi-polymer

microparticles cross-linked with ferric and calcium ions, which

showed superabsorbent behaviour and was considerably stable in

SIF. Their potential applications in drug-delivery systems for con-

trolled and sustained release were further demonstrated by using

BSA as a model protein drug. It is found that their release pro-

file correlated well to their swelling capacity, which can be tuned

by selecting the type of biopolymer, achieving chaotic mixing,

and adjusting the chemical gelation. We believe the biocompati-

ble and biodegradable microparticles with pH stimuli-responsive

behaviour and tunable functions will shed new light in related

fundamental research and applications ranging from medical diag-

nostics to release control.

 Acknowledgements

The authors are thankful to Mr. F. Monti and Dr. P. Tabeling in

ESPCI for fabrication of replica models, to Mr. P. Papineau for kind

assistance in image acquisition software, to Dr. A. Lack for provid-

ing FITC conjugated BSA sample, and to Dr. J.L. Doublier for useful

discussionson the viscosityof alginate solutions.Mr. M. Desprairies

from Cargill is also gratefully acknowledged for alginate samples.

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