synthesis and characterization of uniform-sized hollow chitosan microspheres

Synthesis and Characterizationof Uniform-Sized HollowChitosan Microspheres

SHAN WANG, DEMEI YUDepartment of Applied Chemistry, Xi’an Jiaotong University, Xi’an 710049, Shaanxi Province,People’s Republic of China

Received: January 5, 2009Accepted: May 18, 2009

ABSTRACT: Hollow chitosan (CS) microspheres were obtained using aninverse-emulsion crosslinking method. The temperature and viscosity of the CSemulsion affected the characteristics of the derived microspheres. Themicrospheres were characterized by differential scanning calorimetry, Fouriertransform infrared (FT-IR) spectroscopy, transmission electron microscopy,optical microscopy, scanning electron microscopy (SEM), and laser particle sizeanalysis. FT-IR indicated the sequence of transformations taking place before,during, and after the crosslinking of CS. Optical microscopy and SEM confirmedthe spherical morphology of the gel. The formation of hollow microspheres witha single cavity was identified by SEM. The CS microspheres exhibited a narrowparticle size distribution. Furthermore, particle size could be controlled bychanging the stirring speed. C© 2009 Wiley Periodicals, Inc. Adv Polym Techn28: 40–47, 2009; Published online in Wiley InterScience(www.interscience.wiley.com). DOI 10.1002/adv.20145

KEY WORDS: Chitosan, Crosslinking, Emulsion polymerization, Microgels

Correspondence to: Shan Wang; e-mail: [email protected].

Contract grant sponsor: Shaanxi Province, People’s Republicof China Scholarship Council.

Contract grant number: 08JK482, 08XSYK109.

Advances in Polymer Technology, Vol. 28, No. 1, 40–47 (2009)C© 2009 Wiley Periodicals, Inc.

SYNTHESIS AND CHARACTERIZATION OF UNIFORM-SIZED HOLLOW CHITOSAN MICROSPHERES

Introduction

I nverse-emulsion polymerization is the processtypically used to prepare high-molecular-weight

polymers from water-soluble monomers. The pro-cess involves the emulsification of an aqueous so-lution of the hydrophilic monomer(s) in a nonpolarorganic solvent such as paraffin oil. Surfactant stabi-lizes the emulsion. The scientific literature on inver-sion of emulsions is inconsistent, with exceptions toevery proposed rule.1 It is general to both polymerproducers and end users. Inverse-emulsion poly-merization allows the direct synthesis of the prod-uct without additional purification or drying. For theend user, inverse-emulsion polymers provide a high-solids, easy-to-handle, high-molecular-weight poly-mer product. A key attribute of inverse-emulsionpolymers is the ability to invert the emulsion ondemand, transferring the polymer to the continu-ous phase. The synthesis of polymer microsphereswith hollow interiors has received much attentionin recent years. Because of some unique properties,including low-density, high specific surface area,and good permeation, hollow spheres have poten-tial applications in artificial cells, catalysts, fillers,coatings, pigments, and for the protection of light-sensitive encapsulation and separation components,especially in delivery vehicle systems for the con-trolled release of drugs, cosmetics, inks, and dyes.2

A variety of chemical and physicochemical meth-ods have been reported for the preparation of hol-low materials.3,4 Chitosan (CS) is a cationic polysac-charide derived from a natural polymer, and it isthe second most abundant polysaccharide next tocellulose. It is a biocompatible and biodegradablematerial. Chitosan has been used as a coating ma-terial in pharmaceuticals, a carrier for the immobi-lization of enzymes, and a gel for the entrapmentof cells or microorganisms.5,6 Synthesis of CS mi-crospheres is becoming increasingly important dueto applications in these fields. There are differenttechniques of producing hollow CS microspheres,such as crosslinking, coacervation, emulsification,solvent evaporation, and spray drying.7,8 The size ofdroplets or microspheres is difficult to control andsize distribution is very broad. Therefore, it is nec-essary to prepare uniform-sized microspheres andcontrol the size of the microspheres for their in-tended specific application. In the present investiga-tion, an attempt has been made to synthesize hollowCS microspheres by an inverse-emulsion crosslink-

ing method, a more advantageous process in respectof high yield, short processing time, and the prepa-ration of uniform microspheres. The diameter of theCS particles can be easily controlled in this method(by adjusting the stirring speed). During the prepa-ration, liquid paraffin oil was used as a continuousphase. The effects of CS concentration, type of oilphase and emulsifier, and volume ratio of water tooil phase on the uniformity of the emulsion wereinvestigated. Structural and morphological studiesof the microspheres were carried out using scanningelectron microscopy (SEM) and Fourier transforminfrared (FT-IR) spectroscopy. A combination of ul-trasonic treatment and SEM observation revealedthe hollow structure nature of the microspheres.

Materials and Methods

MATERIALS

Chitosan was purchased from Fluka Co. Ltd.(Buchs, Switzerland). The molecular weight anddeacetylation degree are 70,000 and 55%, respec-tively. Form formaldehyde was obtained fromSigma-Aldrich, Inc. (Munich, Germany). All othermaterials used in the dissolution studies were of an-alytical reagent grade.

PREPARATION OF CS MICROSPHERES

A 250-mL three-necked boiling flask wasequipped with a mechanical stirrer, a nitrogen in-let, and a Hirsch funnel. In the flask, 1.0 g of CSwas dissolved into 20 mL 2% (v/v) aqueous aceticacid. After 10 min, 100 mL of liquid oil and fourdrops of 1% polyvinyl alcohol, which contains anemulsifier (Span-80), were added, dropwise, intothe three-necked flask at 50◦C. The suspension wasthen stirred with a mechanical stirrer at 500 rpmfor 60 min under a nitrogen atmosphere, till thewater phase was uniformly dispersed. A certainamount of 3.7% formaldehyde was slowly addedinto the suspension and stirred at 400 rpm for an-other 30 min at 50◦C. Then, 2 mL of 37% formalde-hyde was added into the suspension, the resultingmixture was stirred continuously under a nitrogenatmosphere for 30 min, NH3 was added to the flaskuntil the pH of the mixture reached 9–10, and thestirring continued for another 15 min. Finally, themixture was filtered, and the filtrate was washed

Advances in Polymer Technology DOI 10.1002/adv 41


consecutively with acetone and then ethanol to re-move the unreacted CS and other impurities, anddried at room temperature. Using this method, itwas possible to prepare hollow CS microspheres ofdifferent uniform sizes by varying the stirring speed.

CHARACTERIZATION OF CSMICROSPHERES

Instruments

The morphology of the CS microspheres was ex-amined by a Philips scanning electron microscope(XL-20), using an accelerating voltage of 15 or 20 kV,and a Hitachi Model H-800 transmission electron mi-croscope at an accelerating voltage of 200 keV. Thesamples were coated with a thin layer of gold be-fore the measurement. FT-IR spectra were recordedon an AVTAR360 Nicolet FT-IR spectrometer usingthe KBr pellet method. Microspheres were sized us-ing a laser particle size analyzer (model MS 1002). Azeta-meter DXD-II electron electrophoresis systeminstrument was used to measure the zeta potential.

Results and Discussion

PREPARATION OF EMULSION

Chitosan was the starting material for thecrosslinking preparation (Fig. 1). The high interfa-cial tension between the “water phase” and the “oilphase” is generally reduced according to necessityby the addition of an amphiphilic surface activeagent or surfactant, that is, a molecule with a polarhead and a nonpolar tail.9 The surfactant moleculesorient themselves according to the polarities of the

CS in HAc

Oil Span-20 Crosslinking

Formaldehyde

Adjust pH

Sonication

CS

CSCS

CSCS

Drying

SEMhollow

CSCSCSCSCS

CSCSCSCSCShollow

CSCS

CSCShollow

CSCSCSCSCS

CSCSCSCSCSHollow

Stirring

FIGURE 1. Schematic for the preparation of chitosan(CS) microsphere. Abbreviations: HAc, acetic acid; SEM,scanning electron microscopy.

involved chemical constituents. Thus, because of thehigh polarity of water, the polar heads of the surfac-tant molecules at the oil–water (o/w) interface areoriented toward the water droplets (Fig. 2). Thesesurfactants are characterized by their hydrophilic–lipophilic balance (HLB) values.9 In an earlier in-vestigation, the HLB values of different nonionicsurfactants in Span-80 exhibited an inverse relation-ship to the average particle size of microspheres.10

The surfactants in the Tween series are basicallythe fatty acid esters of anhydrosorbitols,9 whichare made water soluble by etherifying the freehydroxyl groups with ethylene oxide, for exam-ple, polyoxyethylene sorbitan monooleate (Tween80), polyoxyethylene sorbitan monostearate (Tween60). When they are not etherified, they becomegood oil-soluble emulsifying agents and consti-tute the Span series surfactants, for example, sorbi-tan monooleate (Span-80), sorbitan monopalmitate(Span-40). A high-surfactant HLB value indicates astrongly hydrophilic character, whereas a low valueis an indication of a strong hydrophobic character.9,10

Hence, a nonionic, relatively pH-independent hy-drophobic surfactant, that is, sorbitan monooleate(Span-80), with an HLB value of 4.3 was selectedfor the present study,11−15 in which the hydrophilicsorbitan group acts as the “polar head” and thehydrophobic oleic acid group acts as the “nonpo-lar tail.” Table I presents the characteristics of theparticles of different viscosities. The addition of thesurfactant in amounts of 2.5 g and greater was noteffective for monodisperse sphere formation. Theformation of gel microspheres may be due to theadsorption of the surfactant molecules at the inter-face of the aqueous droplets and the organic solvent,causing a decrease in the interfacial tension, increas-ing the stability of the droplets by steric hindrance,and thereby preventing their coalescence.11

In a study on particle location in emulsion type,Binks and Rodrigues16 indicated that the same par-ticles exhibit a different contact angle with the o/winterface in the two situations and are linked tothe well-documented phenomenon of contact anglehysteresis. They predicted that particles are morehydrophilic and prefer o/w emulsions when ini-tially in water but are more hydrophobic and preferw/o emulsions when initially in oil. So the contin-uous phase of the preferred emulsion becomes thephase in which the particles are first dispersed. Itwas established that preferred emulsions were com-posed of smaller drops compared with nonpreferredemulsions. The unique character of assembled par-ticle aggregates is that the interstices between the

42 Advances in Polymer Technology DOI 10.1002/adv


C

O

H HO

H

H

CH

H

OH

OH

C

H

H

OH

OH+

+

CH2OH

NH

H

CH2

O O

H

HOH

n

n

O

H

H

H

N

OH

CH2OHH O

+OH

n

H2 O

H2 O

O

H

H

H

N H

OH

CH2OHO

nH H

O

H

H

H

H

OH

CH2 OHO

n

NH

O

H

H

H

H

OH

CH2 OHO

n

CH2NH

FIGURE 2. The crosslinking principle of chitosan microsphere.

TABLE ICharacteristics of the Chitosan Microsphere

Run No. Span-80 (g) Acetic Acid (%) Optical Microscopy Results

1 1.5 1 Very little spheres + irregular particles + conglutinate2 1.5 2 Spheres (4�12 μm) + broken spheres + conglutinate3 1.5 3 Mostly spheres (10�120 μm) + some broken spheres + conglutinate4 2 1 Deformed spheres + broken particles6 2 2 Almost all spheres (6�100 μm)7 2 3 Almost all spheres (65�80 μm)8 2.5 1 Very little spheres (5�10 μm) + irregular particles9 2.5 2 Spheres (5�10 μm) + irregular particles + broken spheres10 2.5 3 Spheres (5�60 μm) + broken spheres11 3.0 1 Spheres (10�90 μm) + broken spheres12 3.0 2 All irregular particles13 3.0 3 Deformed spheres + irregular particles + conglutinate



Hea

t f

low

(w

/g)

Temperture (°C )

FIGURE 3. Differential scanning calorimetry of chitosanmicrosphere.

particles form an array of uniform pores, whose sizeis easily adjusted over the nanometer to micrometerscale to control the permeability.17 However, the ad-sorptive particles conglutinated when toluene wasused as the oil-phase solvent.18 The continuous shellhindered the release of packed liquid. Therefore, itwas necessary to choose another oil-phase solvent,whose solubility parameter (δ), polarity, and evenelectrostatic charge property could meet the assem-bled demand and cause minimal swelling of the par-ticles. The relevant properties and the adsorption ofthe particles at the droplet interface are summarizedin Table I. The CS latex particles in their original statecould not be adsorbed at the o/w interface. There-fore, it was necessary to modify their surface proper-ties (i.e., electrostatic charge and hydrophilicity). Theideal modification of the latex surfaces should notcause particle flocculation in the suspension or two-dimensional coagulation after the particles are ad-sorbed on the interface. Furthermore, emulsion-typepreferred drop sizes, and emulsion stability (with re-spect to creaming, sedimentation, and coalescence),were all crucially dependent on the hydrophilicity ofthe particles19 and stirring speed. Different stirringspeeds result in different shear forces, which willproduce different drop sizes in the o/w emulsions.

DIFFERENTIAL SCANNINGCALORIMETRIC ANALYSIS

Microsphere formation was also characterized us-ing differential scanning calorimetry (DSC) (Fig. 3).Under the experimental conditions, the DSC thermo-gram of pure CS shows a sharp endothermic peak

4000 3000 2000 1000 00

5

10

15

20

25

30

35

40

45

50

55

60

b

a

tra

nsp

are

nce

(%

)

Wavelength (cm )

FIGURE 4. Fourier transform infrared spectra ofchitosan (a) and microsphere (b).

at approximately 195◦C.18 The curve shows that thetemperature of the endothermic peak at 75.67◦C isattributed to the volatilization of residual water andorganic solvent. The exothermic peak observed at278.43◦C is related to the decomposition of the poly-mer. This indicates that CS was not present in thesample in the free-form melting of a highly orderedpolymeric structure that has an enthalpy of approx-imately 168.7 J/g. As such, the crosslinking micro-spheres had higher thermal stability when comparedwith the decomposition temperature of CS. Obvi-ously, this result indicates a strong and uniform in-teraction between CS and formaldehyde.

FT-IR SPECTROSCOPIC STUDY

The presence of CS in the microspheres was con-firmed by FT-IR measurements. Curves 1 and 2 inFig. 4 represent the FT-IR spectra of the CS and CSmicrospheres. The FT-IR spectra (4000–400 cm−1) ofthe CS flakes and CS microspheres are shown inFigs. 4a and 4b. Usually, the major peaks of theCS flakes are located at approximately 3400 cm−1

for OH stretching vibration and 1650 cm−1 for NHstretching vibration. The bands observed at 1320and 1380 cm−1 were assigned to CH3 deformationand C N bend. The broad band at 3433 cm−1 wasdue to OH stretching, which overlapped the NHstretching in the same region. The shifts at 1660 and1597 cm−1 were due to amides I and II, respectively.A new peak was observed at 1635 cm−1 for the CSmicrospheres (Fig. 4b), which corresponded to thestretching vibrations of the C N bond, indicatingthe occurrence of the reactions.20



200 m200

50 m

50 m

(a) (b) (c)

FIGURE 5. Optical micrographs of the chitosan microsphere: (a) the water-swollen state; (b) the dry state; and(c) single microsphere in the dry state.

OPTICAL AND SCANNING ELECTRONMICROSCOPY

The morphology of the products was investigatedby optical microscopy and SEM. An optical mi-crograph of water-swollen CS microspheres, whichwere produced by the inverse-emulsion crosslink-ing method with liquid paraffin as the continuousphase, is shown in Fig. 5a. It can be seen that mi-crospheres in the water-swollen state are spheri-cal and have a smooth surface structure. They arenearly monodispersed, and the average diameter isabout 600 μm. The microspheres (not shown) ob-tained from toluene adopted a similar morphology,

but their average diameter was about 300 μm; therewere some deformed spheres and broken particleswhen the continuous phase was toluene. Figure 5bshows typical optical micrograph images of the CSmicrospheres in a dry state. The average diameter isabout 74 μm. There is significant evidence from theoptical micrograph of the hollow structure of the CSmicrospheres (Figs. 5b and 5c). It can also be seenthat microspheres in the dry state are spherical andhave a smooth surface structure.

Figure 6 shows typical SEM images of theCS microspheres under the standard preparativecondition and their enlarged surface structure. Itcan be seen that the microsphere is spherical and

(c) (d)

(a) (b)

6 μm

FIGURE 6. Scanning electron microscopy of the chitosan microsphere: (a) ×100; (b) ×1200; (c) ×1500; and(d) ×10,000.



its diameter is about 74 μm (Fig. 6a). The CS mi-crospheres had regular spherical geometry (Fig. 6b).Further study indicated that the microsphere con-tracts with drying and swells after being immersedin water. Furthermore, it was possible to repeat thecontraction and swelling processes several times (av-erage value = 9 SD). In addition, the microspheresexhibited a narrow particle size distribution. Fig-ure 6c shows a broken CS spherical particle, inwhich the contrast across the diameter reveals thatthe thickness of the shell wall is about 2 μm. Thecomparative analysis of the SEM image in Fig. 6cdemonstrates that the particles are hollow. Figure 6dindicates that CS microspheres exhibit a smoothsurface morphology.

LASER PARTICLE SIZE ANALYSIS ANDTRANSMISSION ELECTRON MICROSCOPYOF CS NANOSPHERES

Figure 7 shows the size distribution of the CS mi-crospheres when the stirring speed increases from1500 to 1800 rpm; the average diameter is 69 nm(Fig. 7a) and 117 nm (Fig. 7b), respectively. The figure

reveals that the microsphere has a very narrow sizedistribution. When the stirring speed was increased,the size of the CS microsphere was smaller becausehigh stirring speed produces a higher shear force,resulting in a relatively low viscosity of the sol-vent, which can be effectively broken into smallerdroplets in the o/w emulsion. Figure 8 shows therecorded representative transmission electron mi-croscopic (TEM) image of the CS microspheres, withan average size of 69 nm. Densely populated, pre-dominantly regularly shaped particles are seen inthe image. It is difficult to find significant direct evi-dence of the hollow structure of the CS microspheresin the TEM image, because the diameter of the micro-spheres is very small. In the TEM micrograph, thereis also the faintest evidence that the CS microspheresare spherical.

PARTICLE SIZE DISTRIBUTION

The CS microspheres observed under the opticalmicroscope indicated that the particle size increasedwith an increase in stirring speed. Under a givenspeed of agitation, the solvent with a relatively

FIGURE 7. The particle size distribution (by number) of chitosan (CS) microsphere: (a) 69 nm; (b) 117 nm.



200 nm

FIGURE 8. Transmission electron microscopy ofchitosan microsphere.

low viscosity can be effectively broken into smallerdroplets, which, after gelation, finally produces par-ticles of a smaller size. Figures 6a and 7 show a typi-cal particle size distribution of the microspheres, in-dicating a very narrow distribution, with an averageparticle size (d) of 74 μm, 117 nm, and 69 nm. Thus,the particle size distribution of CS microspheres canbe easily affected by modifying the stirring speed ofthe solvent.

ZETA POTENTIAL

The measurements of zeta potential were carriedout in a zeta meter DXD-II electron electrophore-sis system instrument using 125 mg of sample(nanoparticles) dispersed in 25 mL of water as pat-tern solution. From this dispersion, 2 mL was dilutedwith 98 mL of distilled water and was used to pre-pare a diluted dispersion with a solid concentrationof 100 ppm. The applied potential was 291 V. Zetapotential of the microemulsion systems gave morestability to nanoparticles (zeta potential was about+27.41 mv (69 nm)), because magnitude of the zetapotential gives an indication of the potential stabilityof the colloidal system. If all the particles in suspen-sion have a large negative or positive zeta potential,then they will tend to repel each other and there isno tendency for the particles to come together. How-ever, if the particles have low zeta potential values,then there is no force to prevent the particles comingtogether and flocculating.

Conclusions

1. Uniform hollow CS microspheres were suc-cessfully prepared by the inverse-emulsioncrosslinking method. Their size can be con-trolled. The particle size distribution of micro-spheres is narrow. The optimum conditions for

the preparation of uniform CS microspheresinclude the following: CS concentration was1.0 wt%; oil phase was liquid paraffin; volumeratio of the water and oil phase was 1:10; andoil emulsifier in oil was 2 g of Span-80.

2. The CS microspheres were characterized byDSC, FT-IR, TEM, optical microscopy, SEM,and laser particle size analysis. The particlesize was controlled by changing the stirringspeed; an increase in stirring speed resulted ina reduction of particle size. Zeta potential wasabout +27.41 mv.

3. TEM, optical microscopy, and SEM confirmedthe spherical morphology of the CS particles.Hollow microspheres with a single cavity wereidentified by SEM.

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