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

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Colloids and Surfaces B: Biointerfaces
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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

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 formed in mineral oil and solidified in the winding microfluidic channels by 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 parameters and their impacts on the swelling behaviour, stability and morphology 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 microparticles were obtained from A–CMC mixture by using ferric chloride as an additional external cross-linking agent. The in situ encapsulation of a model protein, bovine serum 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.

Article history: Received 9 July 2010 Received in revised form 12 August 2010 Accepted 13 August 2010 Available online 21 August 2010 Keywords: Microfluidic synthesis Biopolymer microparticles Rapid mixing In situ encapsulation Smart swelling Controlled release

1. Introduction Since 1990s, with the emerging technology in miniaturisation 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 promoted a microfluidic route to tackle the questions in both fundamentals and applications due to its low cost, low power or reagent consumption and high performance. Specifically, the new developments in microfabrication techniques have enabled the fabrication of very efficient emulsification microstructured devices that allow emulsifying 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 downstream 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:, (A. Fang). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.08.020

tion of the controlled formation of micrometer-sized oil-in-water (O/W) and water-in-oil (W/O) emulsion droplets in a micromachined 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 fabrication of complex microscale geometries enables control over the deformation and breakup of every individual droplet, thus allowing control over the shape, morphology, internal structures [9], chemistry (isotropic and anisotropic/Janus particles) [10–12]. It is the only 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 functional microgels of biopolymers. Pectin and alginate, for example, are known to form complexes with divalent ions, such as Ca2+ , Ba2+ ,

The aqueous liquid phase broke up in continuous oil phase to generate W/O emulsion droplets.0 wt% calcium chloride solution. Cross-linking was induced by rapid mixing of the ingredients inside the droplets while they travel in the winding microchannels downstream. I4. were delivered to I2 and I4. The microchannels have a square cross-section with 100 ␮m by width and 78 ␮m by height. 2. On one hand. Filtered water from a Milli-Q system was used for the preparation of all aqueous solutions. Fang. (a) Immiscible liquids are delivered to microfluidic channels by inlet 1 (I1. The water phase flow was achieved by in situ mixing of three aqueous streams: a biopolymer solution (I2). disperse aqueous phase). which we believe will find wide applications in cosmetics. were able to obtain in situ with the microfluidic devices.1. Winding microchannels (b) without and (c) with additional inlet 6 (I6) is designed for dilution the droplet train with continuous phase. ferric chloride (reagent grade). The microparticles were collected from the microfluidic devices with a large volume buffer solution under gentle agitation. microfluidics has recently emerged as a very promising route to the controlled synthesis of polymeric particles. Briefly.20]. Cathala / Colloids and Surfaces B: Biointerfaces 82 (2011) 81–86 and Sr2+ in aqueous solution. deacetylation ∼70%. respectively. To achieve a bi-biopolymer alginate–CMC (A–CMC) particles. a liquid PDMS prepolymer (in a mixture of 1:10 base polymer:curing agent) was poured onto it. resulting in bas-relief structure from a photoresist SU-8 on silicon wafer which serves as a master for fabricating PDMS molds. BSA. As a rule of thumb. Alginate (A) with molar mass of 94. 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.400 g/mol and sodium carboxymethylcellulose (CMC) with molar mass of 31. calcium chloride (reagent grade). On the other hand. the design of the microstructures was made in a Adobe Illustrator (AI) software and printed out on transparencies as photomask in UV-lithography. continuous oil phase) and inlets 2–4 (I2. we develop a microfluidic route to synthesize and fabricate stimuli-responsive microparticles from naturally occurring polysaccharides. The PDMS was cured at 70 ◦ C for 1 h or more and peeled off the master. respectively.82 A. Due to the versatile control over the local fluids. The biopolymer microparticles are collected from outlet 5 (O5). instead two biopolymer. cosmetics and food industry [19. I3. this microfluidic approach will shed new light in the research for the biocompatible and biodegradable microparticles with stimuli-responsive behaviour. 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. . inlet 3 (I3) and inlet 4 (I4) were supplied with 5 wt% pectin or 4 wt% alginate solution. The emulsified polysaccharide droplets consisting of cross-linking agent were in situ solidified by rapid mixing while traveling in winding microfluidic channels. Materials Pectin (P. The size of the droplets can be controlled by varying the flow rate of the two immiscible liquids. 1. 1. to achieve pectin or alginate microparticles. BSA conjugated with fluorescent dye FITC (BSA-FITC) was synthesized and purified by dialysis in our lab. Finally. bovine serum albumin (BSA). Mineral oil with 2% Span 80 (both from Sigma) was used as continuous oil phase to produce droplet-based emulsion in microfluidic devices. few works have been done by a microfluidic route in the research field of stimuli-responsive biopolymer microparticles toward controlled release. inlet 2 (I2). In this work. Microfluidic 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. material science. those polymers with functional groups such as amino groups and carboxylic acid groups are pH-sensitive [16–18]. shape complexity and size by microfluidic techniques. Typically. To create the PDMS mold. The detailed experimental conditions for the synthesis of each biopolymer microbead sample and its coding information are listed in Table 1. Their smart swelling and release behaviours in different pH conditions were examined and discussed.2. external cross-linking with Fig. pharmaceutics and tissue engineering. Fabrication of PDMS microfluidic devices Procedures for the fabrication of PDMS structures for microfluidics have been described in detail elsewhere [21].1. Small holes were drilled into the PDMS using a borer to produce fluid inlets and outlets. producing the final replica bearing the designed microstructures. surfactants Tween 20 and Span 80 were purchased from Sigma–Aldrich.2. from citrus fruit). Microfluidic fabrication of biopolymer particles Biopolymer microparticles were achieved by emulsifying aqueous solution of biopolymer and its cross-linking agent in mineral oil as the continuous oil phase in the microfluidic devices as shown in Fig. or bi-polymer microparticles encapsulated with a model drug. The synthesis of microparticles from those biopolymers has long been the focus of a very intensive research in biomedicine. bi-polymer microparticles including alginate and carboxymethylcellulose (CMC). B. The optical microscope image is the zoom-in area as indicated by the dotted square in (a). USA). 2. The continuous oil phase was delivered to inlet 1 (I1).2. water and 1. Microfluidic fabrication of microparticles 2. Specifically. alginate and CMC. pharmaceutics. In addition. 2. Together with the ability to control over chemical anisotropy.2. Optical microscope image of microfluidic emulsification and schematic of microfluidic devices used in this work. The two immiscible liquids were supplied to the microchannels using digitally controlled syringe pumps (Harvard Apparatus PHD 2000. its cross-linking agent solution (I4) and inert water (I3). Experimental 2.000 g/mol were generous gifts from Cargill and Aqualon.

A.0 and 1. and in 0. The swelling ratio (SR) has been calculated before as SR% = 100 (Ds − Dd )/Dd . the dried microparticles are often not sphere. those in (b) are I1 = I6 = 2. Fig. however. For release studies or swelling studies. Fang. I2 = I4 = 0. 0.0% CaCl2 1. 1 shows the image of microfluidic emulsification when pectin was used as a model biopolymer. In our case. particles were dispersed under gentle agitation in 0. The amount of BSA in the release medium was analysed by UV spectrophotometry (SPECORD S600.08 mL/h. Sample code Aqueous phase Collecting medium I3 Water Water Water Water Water 1.5% CaCl2 . droplet size can be controlled [25]. respectively. B.5% CMC 0.0% CaCl2 .050 M acetate buffer with 10% CaCl2 .05 mL/h.0. Microfluidic synthesis and in situ encapsulation Due to its ionotropic gelation with Ca2+ and its wide range of applications. Optical microscope images of pectin microparticles fabricated from (a) strait and (b) winding microchannels as shown in Fig. and in 0. 2. In vitro BSA release studies The freshly prepared microparticles encapsulated with BSA were dispersed in 5.03 mL/h. 0.0 mL of sample solution at appropriate intervals was withdrawn and replaced each time by same amount of fresh buffer solution.1.8% A.0 /D1. To study the effect of pH on swelling. I2 = I4 = 0. pH 1. 2a depicts the monodisperse pectin beads with a diameter of 100 ␮m fabricated through the microfluidic device shown in Fig. The cumu- Fig. b Microparticles were collected with a solution of 2% FeCl3 in 0. as simulated gastrointestinal fluid (SGIF). The chemical gelation was controlled by the flow rate ratio of the three aqueous streams. 1a and c. AnalytikJean) at 280 nm. 4% CMC 4% A 4% A 4% A 0.2 denotes the swelling of beads at pH 5. pH 7. RS7. respectively. The swelling behaviour was studied by measuring and comparing the diameters of microparticles when they reached swelling equilibrium.050 M acetate buffer with 3 vol.050 M KH2 PO4 /NaOH solution (pH 7. I3 = 0. pH 5. The unchangeable amount of BSA released in the solution after 48 h can be presumed as the preloading amount of BSA in microparticles [24].0 and D1.5% CMC 1a 1 1 2b 2 1 1 2 2 83 lative protein release was calculated as following: Cumulative release (%) = Amount of released BSA × 100 Amount of preloaded BSA I2 P A A–CMC A/CMC–Fe A–CMC–Fe A–BSA A–CMC–BSA A/CMC–Fe–BSA A–CMC–Fe–BSA 5% P 4% A 4% A 0.1 M HCl/NaCl buffer solution.0). 2.2. where the Ds and Dd are the diameters of dry and swelled particles. 3% Tween 20 (pH 5. As a result. The introduction of a jet of inert water flow sandwiched between pectin and its cross-linking agent solution helps to avoid instant gelation at the cross-junction which may cause unsmooth breakup or even clogging.4. D5. as simulated gastric fluid (SGF).2.5% CaCl2 0.4) at 37 ◦ C using a shaking water bath (50 rpm).5% CaCl2 .5% CaCl2 0.0% CaCl2 1.3. Flow rates used in (a) are I1 = 1.2 are their diameters at pH 5. Scale bar: 50 ␮m. 1a.2 . Cathala / Colloids and Surfaces B: Biointerfaces 82 (2011) 81–86 Table 1 Experimental conditions for synthesis and encapsulation of microparticles on microfluidic devices. I3 = 0. 1.0% BSA 1.0% CaCl2 . Swelling studies of microparticles An Olympus IX51 inverse microscope (Olympus) equipped with a digital camera (Sony. which makes it difficult to measure their size.% Tween 20 (pH 5.0 relative to that at pH 1. Accordingly.50 mL/h. Fig.4/1.5% CMC 1.8% A. we calculated their relative swelling ratio (RS) as following: RS5. pectin was chosen as a model biopolymer to demonstrate the reliability of this microfluidic synthesis approach. respectively [23]. The release assay was reproduced 3 times to obtain the average value.2. Along with varying the flow rate of continuous phase. 0.2 indicates the swelling ratio of microbeads at pH 7. When pectin and divalent Ca2+ solutions are mixed in the emulsion droplet. 0.4. . where RS5.0% BSA 1. Microparticles were extracted and washed with 0. SCD-SX90) was used to acquire images.2 = D5. ferric ions was achieved by collecting the microparticles in the presence of 2% ferric chloride in the buffer solution.5% CMC 0. 3. they were further transferred to different simulated physiological fluids (SPFs). as simulated intestinal fluid (SIF) [22].0/1.4–1.07 mL/h. 4% CMC 4% A The BSA release profiles were plotted as the cumulative amounts in the release medium against the release time.0 mL of 0.0% CaCl2 1.0% BSA I4 1.0 mL/h. 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.0).0/1. The size of the a Microparticles were collected with a solution of 0. 2.050 M potassium acetate/acetic acid.2.050 M acetate buffer with 10% CaCl2 . Results and discussion 3.050 M KH2 PO4 /NaOH.0) by centrifuging the dispersion at 2000 g for 2 min.0% BSA 1. 3% Tween 20 (pH 5.

3a. Smart swelling and controllable release The pH dependence of the swelling of the microparticles was investigated by immersing the microparticles in SPFs at room temperature until a swollen equilibrium was reached. When the gelation rate is superior to the mixing. resulting in irregular shape. leading to the polydispersed beads and poor sphericity. Previous work has demonstrated that.2) and SGIF (pH 5.5%) were reproducibly obtained with a device shown in Fig. Cathala / Colloids and Surfaces B: Biointerfaces 82 (2011) 81–86 microparticles usually falls in the range of 40–100 ␮m from our experiments. which can be attributed to the macropore structures formed by electrostatic repulsion from the highly hydrophilic carboxyl groups in CMC. show obviously increased swelling in SPFs (Fig. Their shape. Confocal fluorescence microscopy images of A–CMC–Fe–BSA hydrogels obtained by using (a) 1. Fang. whereas CMC contributed to the enhanced pore size. a stream of continuous flow was introduced to dilute the droplet train from inlet 6 as shown in Fig.0.4. which could be attributed to the incomplete solidification inside the microchannels. B. 4. diameter more than 100 ␮m). In order to prevent droplet fusion. On the contrary. The latter can be visually spotted by using BSA-FITC instead of BSA as presented in Fig. 3. we introduced CMC into the alginate network for bi-polymer microparticles. And incompletely solidified droplets were easily deformed under agitation when they were collected with a large volume of solution. If large microparticles were preferred (for example. A–CMC–Fe–BSA microparticles using 0. Pectin beads with high monodispersity (c.2 and 5.0 are also visually compared in Fig. The novel design shown in Fig. it is presumed that alginate cross-linked by Ca2+ acted as a strong backbone in the hydrogel structure. To achieve a drug carrier system. Inspired by a recent work from Zhang and coworkers [29]. FITC conjugated BSA was used instead of BSA. inside each droplet.2 values in Fig.0. resulting in poorly integrated beads.0% BSA as a model protein drug was supplied to I3 instead of water. 0. As observed from Fig. showing a totally different structure and morphology from that in Fig. Although the reason is not clear why lower concentration of calcium as cross-linking agent had to be used when encapsulating BSA. 1c. 2a. microparticles except those crosslinked with ferric ions were almost immediately dissolved in SIF at pH 7. When the calcium concentration was further reduced. This is most probably 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.0/1. A–CMC. 4). Surprisingly. In our experiments. 1 including a jet of sandwiched stream will make encapsulation facile simply by replacing the water stream with cell or colloid dispersion solution. The ideal situation is that the chaotic mixing should be much faster than gelation. droplets are solidified before efficient mixing is reached.5% calcium was applied throughout in situ BSA encapsulation.5% CaCl2 as cross-linking agent. leading to polydispersed beads. 4. Therefore. When we applied ferric ions as an additional cross-linking agent for these bi-polymer beads. For all the microparticles. chaotic mixing rate in the winding channel is constant. rapid mixing and chemical gelation take place instantly. a further cross-linking of CMC in A–CMC hydrogels will alter their swelling behaviour. we found that mixing was efficient enough to achieve uniform microparticles as long as the droplets deformed when moving in the winding channels. As we are interested in the fabrication of novel stimuli-responsive functional materials from naturally occurring biopolymers.5 wt% calcium are presented in Fig. Similar to previous work [29]. The in situ mixing can be actually 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 winding channels [26]. a considerable increase in their swelling was achieved. between microparticles cross-linked with . Scale bar: 50 ␮m. Results from Fig. 2b confirm this improvement. To achieve uniform and homogeneous microparticles. 3.0% and (b) 0.5 was observed.0) did not induce any disintegration of the microbeads. the droplet plug should be large enough to “touch” the walls of the channel [27]. in order to achieve chaotic mixing in winding channels. was not uniform. Droplet fusion in large droplet train was often observed. indicating superabsorbent behaviours. As shown in Fig. the flow rates of continuous and disperse phase were adjusted accordingly to achieve large droplets. other conditions remained the same as those to fabricate their BSA-free counterpart as shown in Table 1. The bi-polymer particles. efforts were directed toward microparticles with super swelling properties. At constant flow rate of both disperse and continuous phases.v. which is indicated by their RS5. = 4. this problem can be solved by reducing calcium concentration for a low gelation rate while keeping chaotic mixing rate constant. studied by measuring and comparing the diameters of the swelled microbeads. microparticles with smooth surface but varying shape was achieved (image not shown). prolonged incubation at SGF (pH 1. The swelling of A/CMC–Fe microbeads at pH 1. The zoom-in images of individual beads are also shown. 4. Unless otherwise stated.0/1. however. parts of the beads are missing. Their RS5. the fact that BSA may not be as inert as water should not be neglected. where uniform beads were achieved from rapid mixing by applying the winding microchannels shown in Fig.2 more than 1. As droplets travel downstream. 3b. 1c [28].2. a solution of 1. 1b. Neither pectin nor alginate microparticles demonstrate a tunable smart swelling behaviour. pectin microparticles have the same SR at pH 1. while alginate particles swell slightly more at pH 5. however. 3a.2 and 5. All other conditions are listed in Table 1.84 A. As CMC can be cross-linked by ferric ions [30]. Swelling was Fig.

Cathala / Colloids and Surfaces B: Biointerfaces 82 (2011) 81–86 85 Fig. However. Therefore. their RS5. phase segregation may be dominant. A–CMC. with a RS7. The beads were fully decomposed after 40 h at pH 7. Their full BSA release. We did not observe any disintegration or decomposition in the microbeads after an overnight incubation. At this stage. respectively). 116 h. leading to a minimum uptake of the solvent. we define the swelling capacity as RSm/l = Dm /Dl . The swelling of A/CMC–Fe beads at pH 1. As observed in Fig. Contrary to this rapid release. B. Thus. That A–CMC–Fe or A/CMC–Fe beads have the large swelling capacity in SPFs shows their stability.2 of 1. For the stable A–CMC–Fe microbeads. 4. however. The half release of A/CMC–Fe microbeads was achieved after 5 h. in 1 h for A–CMC microbeads. in A/CMC–Fe microbeads.4. A–CMC–Fe and A/CMC–Fe microbeads show a different release scenario.4. A/CMC–Fe. although the decomposition starts immediately when they were dispersed in the release medium. The maximum swelling is achieved for A–CMC–Fe microbeads in SIF at pH 7. namely. 2. Fig. can be targeted for rapid release. This difference is believed to be directly responsible for their different behaviours in hydration [32].2 . their swelling capacity is in the order of P < A < A–CMC < A/CMC–Fe < A–CMC–Fe. which reached half release after 30 h.0 are also visually compared in right panels. Significant early release was observed.2 . in A–CMC–Fe microbeads. such as epichlorohydrin. Swelling studies of the microparticles in different SPFs. After this initial burst period. especially for the A–CMC–Fe microbeads.0/1. electrostatic repulsions between the ionisable groups are minimum. Among them. a noticeable common phenomenon is that all microbeads demonstrate a rapid burst BSA release at the beginning release stage. .4/1. The relative swelling ratios somehow reflect their swelling behaviours as the pKa of the biopolymers used in this work is larger than 1. where “m” and “l” indicates the most swollen and least swollen states while the microbead still maintains its integrated spherical structure.2.0 and 3. while for other beads. A.4. owing to the surface loaded BSA in hydrogels.4. the BSA release slowed down and was dependent on the decomposition of individ- Fig. This microfluidic approach provides a reliable route to the synthesis of biocompatible microparticles with high control. 4. It is worth mentioning that the bi-polymer beads were formed by a mild cross-linking without involving stringent reactions with hazard reagent. was not observed within 1 week. In spite of their different release rates. For the case of A/CMC–Fe microbeads. For the A–CMC–Fe beads. the full BSA release was reached in a similar sustained way as in A–CMC–Fe microbeads. When alginate and CMC are supplied to microfluidic channels through different inlets and mixing was only carried out in situ chaotically. Scale bar: 50 ␮m. the as-formed A–CMC–Fe microbeads show extreme stability at pH 7. they decomposed after an overnight incubation.2 values reasonably indicate their swelling ability. No attempts were made yet to characterise the microstructures of alginate–CMC bi-polymer beads in more detail. For those microparticles that are unstable at pH 7. The difference here lies in that. Their half release (cumulative release at 50%) was reached in half an hour for A microbeads. Those beads with small swelling capacity. A–CMC. 5. ferric ions but with different methods to mix the two biopolymers. A. for example.4. 4.5. 28. leading to full release of BSA. their full BSA release by final disintegration of microbeads was obtained by prolonged incubation at release medium.0/1.2 (the pKa of alginate and CMC are 4.A. respectively. decomposition of the beads did not start until 2 days in pH 7. A–CMC.4 release medium. It may be closely related to the 3-D structure of the blended bi-polymer networks.8 as shown in Fig. we found the obvious differences in their swelling behaviour and stability in SIF. The dotted lines are guide to eyes. we are not clear why A–CMC–Fe microbeads are more stable than A/CMC–Fe microbeads in SIF. their diameters at pH 1. The periods needed for the A. It is speculated that phase segregation renders the bi-polymer beads more stable at pH 7. 5 shows the BSA in vitro release profiles of the various test microbeads. such as P. The in vitro BSA release profiles from different microparticles in release medium at 37 ◦ C.4/1. indicating potential drugdelivering systems with controlled and sustained release. As a result. RSm/l is equal to their RS7. Their release was much delayed. A–CMC–Fe microbeads to reach 80% release vary as followings: 2. Fang. the CMC and alginate chains remain more intimately mixed.2 provide a good reference to compare their swelling behaviour. RSm/l is their RS5. A and A–CMC microbeads show similar release behaviour. A/CMC–Fe and A–CMC–Fe microparticles. indicated by a change to red color and abrupt increase of BSA in the release medium. The participation of ferric ions in cross-linking is demonstrated by the very distinguishable red color of these microparticles. When alginate and CMC were pre-mixed and supplied to microfluidic channels to synthesize A/CMC–Fe microbeads as detailed in Table 1. 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 networks of CMC and alginate [31].2 and 5. it was obtained over 3 days. in all these hydrogels at pH 1.

[22] C. Anal. T. Nisisako. Xu. E. to Mr. Am. 44 (2005) 724. T.F. [30] C. Chem. Trans. Angew. Li.A.C. Mater. [12] Z. Li. A. Zhang. Song. Seo. Shepherd. [7] J. Y. [16] A. Am. Drumright.K. I. J. Vreeker. B. Gitlin. E. Chem. 46 (2010) 92. M. 2005. de Paula. [9] Z. Angew. Yu. good sphericity and controllable encapsulation. R. T. J.Z. Conclusions In this work. [31] L. Soc. L. Lack for providing FITC conjugated BSA sample. J. achieving chaotic mixing. 127 (2005) 8058. It is found that their release profile correlated well to their swelling capacity. Int.R. Domard. The microbeads achieved by this approach have a narrow size distribution. Y. P. Abdelrahman. Ferrrier. Okushima. and to Dr. Adv. less than 5%. Nisisako. K. G. Mendes. Phillips.M. J. S. Seo. Panizza. J. Nakajima. Ed. Phys. Y. Kikuchi. Chem. J.). Cao.F.P. Moreau. [23] J. Kumachev. T.K. [4] G. W. Appl. 29 (1996) 317. D. H. C. H. B: Biointerfaces 68 (2009) 245. [8] T. Fassihi. Y. Stone. S. [14] D. Dai. Aspects 312 (2008) 24. Nie. R. E. Marquez.A. H. Controlled Release 114 (2006) 1. Pillay. [21] D. Technol. C. A. Li. Food Biophys.v. 4. George.I. Kumacheva. Backov. H. and adjusting the chemical gelation. [25] S. T. Polym. Li. D.T. . Zhuo. Chem. The results clearly reveal that these microbeads show a wide range of pH-sensitive and release behaviours. [19] M. Whitesides.S. Am. We believe the biocompatible and biodegradable microparticles with pH stimuli-responsive behaviour and tunable functions will shed new light in related fundamental research and applications ranging from medical diagnostics to release control. Engl. J. Xu. E. Lyon.D.C. [29] C. Ismagilov. Polym. Weibel. Controlled Release 9 (1999) 243. Acknowledgements The authors are thankful to Mr. P. J. Williams (Eds. Polym. Link. 40 (2007) R319. Soc. Phys. Lewis. M. Denuziere. I. Their sizes ranged from 40 to 100 ␮m in diameter with a c. S. O. Colloids Surf. Whitesides.I. M. J. Zhang. Dietrich. Ed. Lewis. de Carvalho. [27] J. A. Siegwart. Takahashi. D: Appl. D.C. P. Park. Xia.O. Yu. [18] S. which showed superabsorbent behaviour and was considerably stable in SIF. Their potential applications in drug-delivery systems for controlled and sustained release were further demonstrated by using BSA as a model protein drug. Prog. 18 (2006) 1152. Int.A. Semiconductor Lithography: Principles and Materials. 70 (1998) 4974. [17] V.D. Zhang.X. Whitesides.D. thus have application potentials in site-specific controlled and sustained release. Langmuir 20 (2004) 9905. Kawakatsu. [26] M. Dendukuri. Seo. [11] R. Yin. Polym. Cheng. Oil Chem. Int.M. Gao. de Souza. Torii. Chem. J.C. Tice. 1988. Y. Xu. Appelqvist. Doublier for useful discussions on the viscosity of alginate solutions.A. Anna. Christopher. Eng.I. Chang. Feitosa. 2000.E.M.A. L. S.N. Langmuir 24 (2008) 13723. D. to Dr. Liu. Chem.P. Eng. S. Monti and Dr. Zhang. P. Doyle. Binks. Nie. Introduction to Microfluidics. Midoux. Papineau for kind assistance in image acquisition software. which can be tuned by selecting the type of biopolymer. Eur. R. S. W. M. [20] J. R. [10] T. G.A. Garstecki.C. Kumacheva. W.L. Z. Lond. 82 (2001) 584.A. Mater. Philos. Int. Angew. 37 (1998) 551.L. Langmuir 22 (2006) 8618. N. Fang. Abraham. [3] Y. Song. J. B. H. Tabeling. Soc. Desprairies from Cargill is also gratefully acknowledged for alginate samples. Handbooks of Hydrocolloids. Weitz. Ed. and references therein.86 A. Y. Cai. Colloids Surf. Poncin. McDonald. J. P. Trevisan. Carbohydr. A: Physicochem. [32] R. H. We fabricated alginate–CMC bi-polymer microparticles cross-linked with ferric and calcium ions. Gerdts. Jin. [24] L. [13] S. B.S. Fang. A 362 (2004) 1087. New York. Oxford University Press. M. Sci. Mr. Duan. Z. Matyjaszewski. M. 3 (2008) 361. References [1] P. [6] N. J. 48 (2009) 5300. F. J. [2] W. M.J. Cambridge. D. Kim.P. Cathala / Colloids and Surfaces B: Biointerfaces 82 (2011) 81–86 ual microbeads. Yue. Polym. the more sustained release was observed. Hany. The larger RSm/l the microbeads demonstrates.S. R. R.B. E. Liu. J. T. Ismagilov. P.R. G.R. Nie. 58 (2009) 112. B. Nie.M. we describe a microfluidic approach to prepare novel super swelling hydrogels and in situ encapsulation by versatile control over the local fluids and their rapid chaotic mixing in microchannels.J. Adv. Higuchi. Oh. and references therein. J. H. J. Stone. Xiao. Lewis. Food Hydrocolloid 23 (2009) 2278.J. P. Du. Y. 21 (2009) 1. X. Takizawa. [5] T. Plenum. It is found that their release profiles correspond well to their swelling capacity as discussed earlier. 33 (2008) 448.R.M. Nisisako.F. Sci. 74 (1997) 317. S. Kumacheva. Ricardo. Conrad. A.H. X.S. 33 (1997) 1009. [15] G. [28] P. Eur. Yuk. Tabeling in ESPCI for fabrication of replica models. Bringer. Torii. Chem. Rhodes. Z. Langmuir 19 (2003) 127.R. New York. Kumacheva.F. R. Tice. Duffy. M. Schueller. 128 (2006) 9408. Polym. N. S.M. Soc. R. Woodhead Publishing Limited. 31 (2008) 1091. John. A. Langmuir 24 (2008) 13904.

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