Colloids and Surfaces B: Biointerfaces 82 (2011) 8186

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Smart swelling biopolymer microparticles by a microuidic approach: Synthesis, in situ encapsulation and controlled release
Aiping Fang , Bernard Cathala
BIA-NANO, INRA, Rue de la Graudire, 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 microuidic 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 solidied in the winding microuidic 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 microuidic 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, ACMC); (2) rapid mixing; (3) concentration and type of cross-linking agent. Superabsorbent microparticles were obtained from ACMC 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 uids. Compared with alginate and ACMC microparticles cross-linked with calcium ions, ACMC microparticles cross-linked with both calcium and ferric ions demonstrate a signicantly delayed release. The controllable release prole, the facile encapsulation as well as their biocompatibility, biodegradability, mucoadhesiveness render this microuidic approach promising in achieving biopolymer microparticles as protein drug carrier for site-specic 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: Microuidic synthesis Biopolymer microparticles Rapid mixing In situ encapsulation Smart swelling Controlled release

1. Introduction Since 1990s, with the emerging technology in miniaturisation and MEMS, microuidics has been considerably diversied for widespread applications in the multidisciplinary elds of chemistry, biology and physics [1]. Microfabrication by silicon technologies [2] and the recent soft lithography [3] has greatly promoted a microuidic route to tackle the questions in both fundamentals and applications due to its low cost, low power or reagent consumption and high performance. Specically, the new developments in microfabrication techniques have enabled the fabrication of very efcient emulsication microstructured devices that allow emulsifying a uid in another immiscible uid [4]. Thus, droplets [5] or bubbles [6,7] can be continuously produced and dispersed in a continuous uid owing within these microuidic devices. If the as-generated droplets or bubbles can be solidied downstream either thermally or chemically, one achieves synthesis of microparticles by a microuidic route. In fact, continuous efforts have been dedicated to this approach since the rst demonstra-

Corresponding author. Tel.: +33 240675068; fax: +33 240675043. E-mail addresses: aiping.fang@nantes.inra.fr, aiping.fang@gmail.com (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 microuidic fabrication approach stem from the possibility to generate highly monodisperse droplets (the coefcient 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 ow eld 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) [1012]. 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+ ,

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and Sr2+ in aqueous solution. In addition, those polymers with functional groups such as amino groups and carboxylic acid groups are pH-sensitive [1618]. 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, microuidics 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 microuidic route in the research eld of stimuli-responsive biopolymer microparticles toward controlled release. In this work, we develop a microuidic route to synthesize and fabricate stimuli-responsive microparticles from naturally occurring polysaccharides. Microuidic devices from poly(dimethylsiloxane) (PDMS) with versatile control over the local uids and their mixing in microchannels by complex microscale geometries were developed for the fabrication of polysaccharide microparticles. The emulsied polysaccharide droplets consisting of cross-linking agent were in situ solidied by rapid mixing while traveling in winding microuidic channels. Due to the versatile control over the local uids, bi-polymer microparticles 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 microuidic devices. Their smart swelling and release behaviours in different pH conditions were examined and discussed. Together with the ability to control over chemical anisotropy, shape complexity and size by microuidic techniques, this microuidic approach will shed new light in the research for the biocompatible and biodegradable microparticles with stimuli-responsive behaviour, which we believe will nd wide applications in cosmetics, 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, surfactants Tween 20 and Span 80 were purchased from SigmaAldrich. Alginate (A) with molar mass of 94,400 g/mol and sodium carboxymethylcellulose (CMC) with molar mass of 31,000 g/mol were generous gifts from Cargill and Aqualon, respectively. BSA conjugated with uorescent dye FITC (BSA-FITC) was synthesized and puried by dialysis in our lab. Filtered water from a Milli-Q system 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 microuidic devices.

2.2. Microuidic fabrication of microparticles 2.2.1. Fabrication of PDMS microuidic devices Procedures for the fabrication of PDMS structures for microuidics have been described in detail elsewhere [21]. Briey, 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 photoresist SU-8 on silicon wafer which serves as a master for fabricating PDMS molds. To create the PDMS mold, a liquid PDMS prepolymer (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 nal replica bearing the designed microstructures. Small holes were drilled into the PDMS using a borer to produce uid inlets and outlets. Finally, PDMS devices were formed by irreversibly bonding the PDMS replica with a at PDMS slab (in a mixture of 1:20 base polymer:curing agent) at 70 C for 2 h or more. 2.2.2. Microuidic 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 microuidic 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 delivered to inlet 1 (I1). The water phase ow 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 1.0 wt% 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 controlled by varying the ow 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 alginateCMC (ACMC) particles, instead two biopolymer, alginate and CMC, were delivered to I2 and I4. The microparticles were collected from the microuidic devices with a large volume buffer solution under gentle agitation. The detailed experimental conditions for the synthesis of each biopolymer microbead sample and its coding information are listed in Table 1. Specically, external cross-linking with

Fig. 1. Optical microscope image of microuidic emulsication and schematic of microuidic devices used in this work. (a) Immiscible liquids are delivered to microuidic channels by inlet 1 (I1, continuous oil phase) and inlets 24 (I2, I3, I4, disperse aqueous phase). The biopolymer microparticles are collected from outlet 5 (O5). Winding microchannels (b) without and (c) with additional inlet 6 (I6) is designed for dilution the droplet train with continuous phase. The microchannels have a square cross-section with 100 m by width and 78 m by height. The optical microscope image is the zoom-in area as indicated by the dotted square in (a).

A. Fang, B. Cathala / Colloids and Surfaces B: Biointerfaces 82 (2011) 8186 Table 1 Experimental conditions for synthesis and encapsulation of microparticles on microuidic devices. Sample code Aqueous phase Collecting medium I3 Water Water Water Water Water 1.0% BSA 1.0% BSA 1.0% BSA 1.0% BSA I4 1.0% CaCl2 1.0% CaCl2 1.0% CaCl2 , 0.5% CMC 1.0% CaCl2 1.0% CaCl2 , 0.5% CMC 0.5% CaCl2 0.5% CaCl2 , 0.5% CMC 0.5% CaCl2 0.5% CaCl2 , 0.5% CMC 1a 1 1 2b 2 1 1 2 2

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lative protein release was calculated as following: Cumulative release (%) = Amount of released BSA 100 Amount of preloaded BSA

I2 P A ACMC A/CMCFe ACMCFe ABSA ACMCBSA A/CMCFeBSA ACMCFeBSA 5% P 4% A 4% A 0.8% A, 4% CMC 4% A 4% A 4% A 0.8% A, 4% CMC 4% A

The BSA release proles were plotted as the cumulative amounts in the release medium against the release time. 3. Results and discussion 3.1. Microuidic 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 demonstrate the reliability of this microuidic synthesis approach. Fig. 1 shows the image of microuidic emulsication 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 100 m fabricated through the microuidic device shown in Fig. 1a. The introduction of a jet of inert water ow 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. The chemical gelation was controlled by the ow rate ratio of the three aqueous streams. Along with varying the ow rate of continuous phase, droplet size can be controlled [25]. The size of the

a Microparticles were collected with a solution of 0.050 M acetate buffer with 10% CaCl2 , 3% Tween 20 (pH 5.0). b Microparticles were collected with a solution of 2% FeCl3 in 0.050 M acetate buffer with 10% CaCl2 , 3% Tween 20 (pH 5.0).

ferric ions was achieved by collecting the microparticles in the presence of 2% ferric chloride in the buffer solution. Microparticles were extracted and washed with 0.050 M 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 uids (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 comparing 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 uid (SGF), and in 0.050 M potassium acetate/acetic acid, pH 5.0, as simulated gastrointestinal uid (SGIF), and in 0.050 M KH2 PO4 /NaOH, pH 7.4, as simulated intestinal uid (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, the dried microparticles are often not sphere, which makes it difcult 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 7.41.2. 2.4. In vitro BSA release studies The freshly prepared microparticles encapsulated with BSA were dispersed in 5.0 mL of 0.050 M KH2 PO4 /NaOH solution (pH 7.4) at 37 C using a shaking water bath (50 rpm). 1.0 mL of sample 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 spectrophotometry (SPECORD S600, AnalytikJean) at 280 nm. 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-

Fig. 2. Optical microscope images of pectin microparticles fabricated 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 = 0.08 mL/h, those in (b) are I1 = I6 = 2.0 mL/h, I2 = I4 = 0.03 mL/h, I3 = 0.07 mL/h. Scale bar: 50 m.

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microparticles usually falls in the range of 40100 m from our experiments. Pectin beads with high monodispersity (c.v. = 4.5%) were reproducibly 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 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. The in situ mixing can be actually improved by including winding channels due to the fact that chaotic advection for efcient mixing is generated by the unsteady uid ow inside each droplet when it travels through the winding channels [26]. Results from Fig. 2b conrm this improvement, where uniform beads were achieved from rapid mixing by applying 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 efcient enough to achieve uniform microparticles as long as the droplets deformed when moving in the winding channels. If large microparticles were preferred (for example, diameter more than 100 m), the ow rates of continuous and disperse phase were adjusted accordingly to achieve large droplets. Droplet fusion in large droplet train was often observed, leading to polydispersed beads. In order to prevent droplet fusion, a stream of continuous ow was introduced to dilute the droplet train from inlet 6 as shown 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 protein drug was supplied to I3 instead of water. Unless otherwise stated, other conditions remained the same as those to fabricate their BSA-free counterpart as shown in Table 1. As droplets travel downstream, inside each droplet, rapid mixing and chemical gelation take place instantly. The ideal situation is that the chaotic mixing should be much faster than gelation. When the gelation rate is superior to the mixing, droplets are solidied before efcient 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 ow rate of both disperse and continuous phases, chaotic mixing rate in the winding channel is constant. Therefore, this problem can be solved by reducing calcium concentration for a low gelation rate while keeping chaotic mixing rate constant. ACMCFeBSA microparticles using 0.5 wt% calcium are presented in Fig. 3b, showing a totally different structure and morphology from that in Fig. 3a. When the calcium concentration was further reduced, microparticles with smooth surface but varying shape was achieved (image not shown), which could be attributed to the incomplete solidication inside the microchannels. And incompletely solidied droplets were easily 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 concentration 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 temperature until a swollen equilibrium was reached. Swelling was

Fig. 3. Confocal uorescence microscopy images of ACMCFeBSA hydrogels obtained by using (a) 1.0% and (b) 0.5% CaCl2 as cross-linking agent. All other conditions 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: 50 m.

studied by measuring and comparing the diameters of the swelled microbeads. 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 microparticles 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, ACMC, show obviously increased swelling in SPFs (Fig. 4), which can be attributed to the macropore structures formed by electrostatic repulsion from the highly hydrophilic carboxyl groups in CMC. Similar to previous work [29], it is presumed that alginate cross-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 ACMC hydrogels will alter their swelling behaviour. When we applied ferric ions as an additional cross-linking agent for these bi-polymer beads, a considerable increase in their swelling was achieved. Their RS5.0/1.2 more than 1.5 was observed, indicating superabsorbent behaviours. The swelling of A/CMCFe 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 crosslinked 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 the microparticles in different SPFs. The swelling of A/CMCFe beads at pH 1.2 and 5.0 are also visually compared in right panels. Scale bar: 50 m.

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 supplied to microuidic channels to synthesize A/CMCFe microbeads as detailed in Table 1, they decomposed after an overnight incubation. When alginate and CMC are supplied to microuidic channels through different inlets and mixing was only carried out in situ chaotically, the as-formed ACMCFe microbeads show extreme stability 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 networks 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 ACMCFe microbeads are more stable than A/CMCFe 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 ACMCFe microbeads, phase segregation may be dominant; in A/CMCFe microbeads, the CMC and alginate chains remain more intimately mixed. This difference is believed to be directly responsible for their different behaviours in hydration [32]. No attempts were made yet to characterise the microstructures of alginateCMC bi-polymer beads in more detail. It is speculated that phase segregation renders the bi-polymer beads more stable at pH 7.4. The relative swelling ratios somehow reect their swelling behaviours as the pKa of the biopolymers used in this work is larger than 1.2 (the pKa 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 ACMCFe microbeads in SIF at pH 7.4, with a RS7.4/1.2 of 1.8 as shown in Fig. 4. Therefore, we dene 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 ACMCFe 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 < ACMC < A/CMCFe < ACMCFe. That ACMCFe or A/CMCFe beads have the large swelling capacity in SPFs shows their stability, indicating potential drugdelivering systems with controlled and sustained release. Those beads with small swelling capacity, such as P, A, ACMC, can be targeted for rapid release. It is worth mentioning that the bi-polymer beads were formed by a mild cross-linking without involving stringent reactions with hazard reagent, such as

epichlorohydrin. This microuidic approach provides a reliable route to the synthesis of biocompatible microparticles with high control. Fig. 5 shows the BSA in vitro release proles of the various test microbeads, namely, A, ACMC, A/CMCFe and ACMCFe microparticles. Among them, A and ACMC microbeads show similar release behaviour. Signicant early release was observed. Their half release (cumulative release at 50%) was reached in half an hour for A microbeads, in 1 h for ACMC microbeads. The beads were fully decomposed after 40 h at pH 7.4, leading to full release of BSA. Contrary to this rapid release, ACMCFe and A/CMCFe microbeads show a different release scenario. Their release was much delayed, especially for the ACMCFe microbeads, which reached half release after 30 h. The half release of A/CMCFe microbeads was achieved after 5 h, their full BSA release by nal disintegration of microbeads was obtained by prolonged incubation at release medium. However, it was obtained over 3 days. For the stable ACMCFe 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/CMCFe microbeads, although the decomposition starts immediately when they were dispersed in the release medium, the full BSA release was reached in a similar sustained way as in ACMCFe microbeads. The periods needed for the A, ACMC, A/CMCFe, ACMCFe 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 and was dependent on the decomposition of individ-

Fig. 5. The in vitro BSA release proles from different microparticles in release medium at 37 C. The dotted lines are guide to eyes.

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ual microbeads. It is found that their release proles 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 range of pH-sensitive and release behaviours, thus have application potentials in site-specic controlled and sustained release. 4. Conclusions In this work, we describe a microuidic approach to prepare novel super swelling hydrogels and in situ encapsulation by versatile control over the local uids and their rapid chaotic mixing in microchannels. The microbeads achieved by this approach have a narrow size distribution, good sphericity and controllable encapsulation. Their sizes ranged from 40 to 100 m in diameter with a c.v. less than 5%. We fabricated alginateCMC 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 controlled and sustained release were further demonstrated by using BSA as a model protein drug. It is found that their release prole 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 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. 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 providing FITC conjugated BSA sample, and to Dr. J.L. Doublier for useful discussions on the viscosity of alginate solutions. Mr. M. Desprairies from Cargill is also gratefully acknowledged for alginate samples.

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