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wileyonlinelibrary.com106 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Healthcare Mater.2012, 1, 106111

Jian-Hong Xu,* Hong Zhao, Wen-Jie Lan, and Guang-Sheng Luo*

A Novel Microfluidic Approach for MonodispersedChitosan Microspheres with Controllable Structures

Prof. J.-H. Xu, H. Zhao, W.-J. Lan, Prof. G.-S. LuoThe State Key Lab of Chemical EngineeringDepartment of Chemical EngineeringTsinghua University100084, Beijing, P. R. ChinaE-mail: [email protected];[email protected]

DOI: 10.1002/adhm.201100014

Chitosan, obtained by alkaline deacetylation of chitin, whichis the second-most abundant polysaccharide next to cellulose,is the only base polysaccharide in nature and has outstandingproperties of nontoxicity, biocompatibility, biodegradability,and low cost. Chitosan has one primary amino and two freehydroxyl groups for each C6unit. This, in turn, makes chitosana weak base and insoluble either in water or in organic solvents.However, it is soluble in dilute aqueous acidic solution. In therecent years, as a new type of functional materials, chitosan has

great potential application in adsorption and isolation of pro-tein, catalytic carrier, enzyme immobilization, and controlleddrug release in the form of fibers, membranes, microspheres,and capsules.[1]

Chitosan microspheres play an important role because oftheir special size and structures.[2]While the control of chitosanparticles size and size distribution are very important to theirsuccessful biomedical applications,[3]Wang et al.[4]pointed outthat the microspheres should be defined in a specific range,usually bellow 60 m, and have uniform size while used asthe microreactors in the field of organism catalysis and as thedrug carriers. Various methods are presently available to pre-pare chitosan microspheres, for example, a emulsification-curing method, simple coacervation, complex coacervation,

etc.[5]Although the above-mentioned techniques are feasible forpreparation, there are considerable drawbacks such as unstableyield, tedious procedures, non-uniform particle sizes with awide size distribution, and lack of process repeatability. It hasbecome imperative for the pharmaceutical industry to developa reproducible method for generating chitosan microsphereswith uniform particle size in a controlled manner, especiallywith size less than 60 m. On the other hand, the control of themicrospheres structures is shown to affect the release rate andthe drug dosage, which is also important to their application asdrug carriers. Dambies et al.[6]prepared a double-layer structurecorresponding to a very compact 100-m-thick external layerand an internal structure of small pores by preparing the chi-

tosan gel beads in a molybdate solution under optimum condi-tions. Wei et al.[7]prepared the chitosan microspheres with four

different structures by modifying chitosan in different ways. Tothe best of our knowledge, there is no integrated method to pre-pare chitosan microspheres with different structures at present.Each structure has its own method to prepare and some of themethods are usually complicated.

Recently, microfluidic methods have been developed asthe novel approaches for the controllable synthesis of mono-dispersed microbeads. Highly monodispersed droplets withnarrow size distribution and spherical polymeric microparti-

cles can be obtained by using microfluidic methods.[

8

]

Severalresearch groups[9]have attempted to utilize such techniques toproduce chitosan microspheres, and monodispersed chitosanmicrospheres with controlled sizes (from 100 to 500 m diam-eter) and a narrow size distribution (a variation of less than10%) were synthesized successfully.

The requirements for the chitosan microspheres vary signifi-cantly from case to case in biomedical and biocatalysis appli-cations. In clinical therapy, it is necessary to maintain proteindrugs as a specific therapeutic serum concentration. Then themicrospheres with the release profile of a minimal initial burstand a relatively well-controlled release are likely to be ideal car-riers for these protein drugs. On the contrary, microspheres witha strong initial burst are suitable for treating cancer or hepatitis,

which need short but intensive administration of drugs. [7a] Inthe field of biocatalysis, the enzyme should has good stability inthe microspheres. Therefore, it is very important to develop anintegrated and simple approach to prepare the chitosan micro-spheres with relatively small size (

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Figure 1. a) Flow chart of the experiments. b) Optical/Fluorescent micrographs of droplets andmicrospheres with different continuous phase flow rate at the fixed dispersed flow rate of 5.0L min1.1) Droplets,Qc=400L min

1; 2) microspheres,Qc=400L min1; 3) droplets,Qc=1550L min

1;4) microspheres, Qc=1550 L min

1. c) The microspheres with different size by changing thesolidification time at the fixed flow rates of Qc=400 L min

1and Qd=5.0 L min1. d) The

effect of the continuous phase flow rate on the size of the microspheres with a solidificationtime of 35 min at a fixed dispersed flow rate of 5.0 L min1.

the n-octanol in the solidification bath when the droplets wereshaken in the solidification bath. Therefore, the droplets gradu-ally solidified from the surface to the inside with the residencetime increasing, and monodispersed chitosan microspherescould be prepared.

The formed microspheres were highly monodispersed, andthe microspheres size was mainly controlled by the contin-uous flow rate and solidification time, as shown in Figure 1 .The chitosan droplets and microspheres decreased sharply byincreasing the continuous flow rate (Qc) (Figure 1b). The dia-meter of the microspheres changes from 130 m to 66 m with

increasing solidification time at a fixed flowrate (Figure 1c). The average diameter of themicrospheres solidified for 35 min changesfrom 66 m to 25 m with the increasingcontinuous phase flow rate (Figure 1d). Sowe successfully prepared monodispersed

chitosan microspheres with relatively smallsize by using a novel and simple approach.The microspheres size could be controlledfrom 130 m to 25 m simply by changingthe continuous flow rate and solidificationtime.

The control of the microspheres struc-tures could also be well accomplished byregulating solidification time. In the pastfew decades, chitosan microspheres havealways been prepared by facile chemicalcrosslinking using glutaraldehyde (CGmicrospheres) or solvent extraction method(SG microspheres).[7,9] Previous studies

have demonstrated that CG microspheresusually have a solid or coreshell structurewithout any pores on their surface,[7,9a9e]while SG microspheres exhibit porous andcoreshell structures both on the surface andon the inside.[9f,9g] Thus, in an attempt tofurther control the structure of the chitosanmicrospheres, we attempted to develop a newpreparation approach combining the abovetwo methods. We can call the chitosan micro-spheres prepared using this novel methodCSG microspheres. Scheme 1 showsthe mechanism of the microsphere struc-

tures generation. The chemical crosslinkingreaction between glutaraldehyde and chi-tosan and water extraction could occur in asimultaneous way. Therefore, the size of themicrospheres decreased and the structurechanged with increasing solidification time.The forming process of CSG microspheresinvolves three main stages with the followingtransformation of the microsphere structures:1) First, the water in the droplets wasextracted out, which forms pores at themicrospheres surface, allowing the porousstructure to distribute no matter on thesurface or in the inside. We named them

CSGA microspheres. 2) With increasingsolidification time, the chemical crosslinking taking placebetween aldehydic group and amino group can form dense struc-ture on the surface, forming a coreshell structure with a solidshell and porous core. We named those CS-G-B microspheres.3) Diffusion of glutaraldehyde molecules into the inside of themicrospheres and crosslinking with the chitosan moleculesresults in a thicker and thicker solid shell layer, which finallyleads to a complete solid structure. We named them CSGCmicrospheres. In other words, the structures of the micro-spheres can be easily controlled by simply changing the solidi-fication time.

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Figure 2 shows the characterization results of the CSGAmicrospheres that were prepared under the experimental condi-tions of Qc=400 L min

1, Qd=5.0 L min1, and the solidifi-

cation time of 10 min. From the SEM images, the microspheresare porous both on the surface and on the inside (Figure 2ato c). The pores are mainly caused by the extraction of waterfrom the microspheres to the solidification bath.[9f] Wei et al.have demonstrated the CG microspheres show remarkableautofluorescent properties that have been attributed to the n

transitions of C=N bonds in the Schiff bases formed during thecrosslinking reaction.[7]This autofluorescent properties providea way to characterize the thickness of the crosslinking layer ofCSG microspheres by using laser scanning confocal micro-scopy (LSCM), as shown in Figure 2d. The crosslinking layerattains a thickness of only a few micrometers at a solidifica-tion time of less than 10 min. A gradual decrease of the size

of the surface pores and the growth of the shell thickness ofthese porous microspheres have been obtained by increasingthe solidification time.

As the solidification time was increased to 20 min, thechemical crosslinking taking place between the aldehydic groupand the amino group formed dense structures on the surface,allowing the microspheres to form a coreshell structure with asolid shell and a porous core. CSGB microspheres were suc-cessfully prepared (Figure 2eh). The thickness of the denseshell is mainly affected by the diffusion of water from micro-spheres into the solidification bath and the diffusion of glutar-aldehyde molecules into the inside of the microspheres andcrosslinking with the chitosan molecules. A gradual growth ofthe dense shell thickness of these CSGB microspheres could

be obtained by increasing the solidification time.Given a long enough solidification time (about 35 min),

glutaraldehyde molecules diffused into the core of the micro-spheres, and the crosslinking reaction occurred everywherein the microspheres. CSGC microspheres with a solidstructure were formed (Figure 2il). The size and structureof the microspheres would no longer change with increasingsolidification time, which can be seen from the comparison ofFigure 2k and l with Figure S5 in the Supporting Information.The crosslinking reaction is structurally homogeneous insidethe microspheres, which could enhance the mechanical sta-bility of the microspheres.

From the above results, three typicalkinds of chitosan microspheres have beensuccessfully prepared by changing only thesolidification time from 10 to 35 min, usinga novel method. Compared to traditionalprocedures, the method presented here has

enabled the preparation of monodispersedmicrospheres with low coefficients of varia-tion (CV) (

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Figure 2. a) Scanning electron microscopy (SEM) image of CSGA microspheres. b) SEM image of the surface structure of CS-G-A microspheres.c) SEM image of the inner structure of CSGA microspheres. d) Laser scanning confocal microscopy (LSCM) image of CSGA microspheres.e) SEM image of CSGB microspheres. f) SEM image of the surface structure of CSG B microspheres. g) SEM image of the inner structure of CSGBmicrospheres. h) LSCM image of CSGB microspheres. i) SEM image of CSGC microspheres. j) SEM image of the surface structure of CSGB micro-spheres. k) SEM image of the inner structure of CSGC microspheres with the solidification time of 35 min. l) LSCM image of CSGC microsphereswith the solidification time of 35 min. The scale bars represent 200 m in (a), 500 m in (e,i), 100 m in (d,h,l), and 50 m in (b,c,f,g,j,k).

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average diameter from 135 to 55 m were formed by controllingthe flow rate of the continuous phase via a capillary-embeddedT-junction microfluidic device. The aqueous droplets formedwere solidified by combining chemical crosslinking with sol-

vent extraction. Microspheres with three typical structures,namely porous, coreshell, and solid structures, were success-fully prepared by controlling the solidification time. The sizeof the chitosan microspheres could be controlled from 130 mto 25 m and it further reduced to a few micrometers withdecreasing capillary diameter and increasing continuous flowrate. Furthermore, the results have shown that microsphereswith different structures showed different release velocitiesin conrolled drug release and that in situ lipase immobilizedmicrospheres with compact external layer had about 10 timeshigher activity than free lipases and showed a lower enzymelose rate. Therefore, the monodispersed chitosan microspheres

with controllable size discussed here are promising systemswith better reproducibility, more repeatable release behavior,higher bioavailability, and passive targetability. The tunabilityof microspheres structure enables the use of these systems toaddress specific requirements for use as protein drug carriersand biocatalysis.

Experimental Section

Materials: Chitosan (0.2 g) with an average molecular weight of180 kD (purchased from Yuhuan Ocean Biochemical Co., Ltd., Zhejiang,P. R. China) was dissolved in acetic acid (0.2 g, purchased from VASChemical Co., Ltd., Tianjin, P.R. China) to prepare a polymer aqueoussolution (10 g). The aqueous phase was used as the dispersed phase inour experiments to form monodispersed droplets. Span80 (2 g) dissolvedin n-octanol (100 g, all purchased from VAS Chemical Co., Ltd., Tianjin,P. R. China) was used as the continuous phase. Glutaraldehyde (0.06 g,purchased from VAS Chemical Co., Ltd., Tianjin, P. R. China) and Span80(0.24 g, dissolved in n-octane (12 g) was used as the solidification bathand glutaraldehyde was used as the crosslinking reagent.

Experimental Microfluidic Device: The microfluidic device wasfabricated on two 40 mm 20 mm 3 mm polymethyl methacrylate(PMMA) plates using micromachining technology. A Teflon tube with0.5 mm inner diameter was inserted as the continuous phase inletand the multiphase flow channel, while a Teflon tube with 0.05 mminner diameter was inserted as the dispersed phase inlet (Figure S1in the Supporting Informaiton). The microfluidic device was obtainedby sealing the two PMMA plates together. Two microsyringe pumpsand two gastight microsyringes were used to pump the fluids intothe microfluidic device. The droplets forming in the Teflon tube werecollected with a solidification bath placed on a shaker.

Preparation of Monodispersed Chitosan Microspheres: In a typicalpreparation experiment, the aqueous solution with 2 wt% chitosan and2 wt% acetic acid was served as the dispersed phase, which is injectedinto the microchannel and separated into monodispersed droplets bythe shear force of the continuous flow. An n-octanol solution with 2 wt%

Span80 was used as the continuous phase. 0.5 wt% glutaraldehyde and2 wt% Span80 added into n-octane was used as the solidification bath.The Schiffs base reaction between gluraltadehyde and chitosan and theextraction of water by n-octanol were employed to solidify the dropletsin the solidification bath. The microspheres with different solidificationtime were obtained by controlling the time droplets shaken in thesolidification bath. Finally, the spheres were washed with n-octane anddried by freeze-drying.

In situ Preparation of BSA-Loaded and Lipases-Loaded ChitosanMicrospheres: BSA is used as the protein drug model in our experiment.The continuous phase and the solidification bath were as same asthese described above. Besides the component of the dispersed phase,0.2 wt% BSA was added and, at the same time, the quantity of aceticacid was reduced from 2 wt% to 0.33 wt% in order to avoid the BSAlosing its activity. The dispersed phase should be used after centrifuging.The formation of the microspheres is identical to the process presented

above. The lipase-coated in situ chitosan microspheres can be obtainedby putting the same quantity of lipase into the dispersed phase insteadof the BSA.

Characterization: Droplets and microspheres were observed withan optical microscope (Olympus) and an on-line CCD (Pixlink). Moredetailed structures were observed using scanning electron microscopy(SEM, FEI XL30). Fluorescence was observed using laser sanningconfocal microscopy (LSM710, Zeiss). The BSA/lipases-loaded sphereswere characterized by desorption of BSA and the enzyme activity oflipases. The BSA release experiment was performed by putting theBSA-coated in situ chitosan microspheres into the phosphoric acidbuffer solution (pH = 7.0, 0.05 M), shaking from 1.0 h to 100 h, andthen determining the BSA concentration of the solution using UVvisspectrophotometer. [7] The standard enzyme activity experiment in

Figure 3. a) In vitro BSA release profiles measured for the different typesof microspheres. b) The relationship of enzyme activity and cycle usingtimes.

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aqueous media is the hydrolyzation reaction of acetic acid glyceride in30 min.

Supporting Information

Supporting Information is available from the Wiley Online Library orfrom the author.

Acknowledgements

This work was supported by the National Natural Science Foundation ofChina (21036002, 21136006, 20806042) and A Foundation for the Authorof National Excellent Doctoral Dissertation of PR China (FANEDD201053).

Received: October 29, 2011Published online: December 14, 2011

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