2013

Journal of Microencapsulation, 2013; 30(5): 498–509� 2013 Informa UK Ltd.ISSN 0265-2048 print/ISSN 1464-5246 onlineDOI: 10.3109/02652048.2012.758179

Microencapsulation of menthol by crosslinked chitosan via porousglass membrane emulsification technique and their controlledrelease properties

Roongkan Nuisin1, Jaruwan Krongsin2, Supaporn Noppakundilograt3

and Suda Kiatkamjornwong3,4

1Department of Environmental Science, Faculty of Science, Chulalongkorn University, Bangkok, Thailand,2Multidisciplinary Program of Petrochemistry and Polymer Science, Faculty of Science, Chulalongkorn University,Bangkok, Thailand, 3Department of Imaging and Printing Technology, Faculty of Science, Chulalongkorn University,Bangkok, Thailand, and 4The Royal Institute of Thailand, Sanam Sueaba, Dusit, Bangkok, Thailand

AbstractChitosan-encapsulated menthol microcapsules were successfully prepared in an oil-in-water (o/w) emulsionusing the Shirasu Porous Glass (SPG) membrane emulsification technique and high-speed dispersion tech-nique for preparing a mixed o/w emulsion. The size of the menthol-loaded chitosan microcapsules wasstrongly depended on the average pore size of the SPG membrane and the amount of menthol loading inthe dispersed phase. The membrane pore size of 5.2 mm was suitable for a viscous dispersed phase con-taining light mineral oil. The average diameter of emulsion droplets of 28.3 mm was obtained. Increasing thementhol loading in the dispersion phase from 5% to 10% w/w of chitosan decreased the emulsion dropletsize with a broad size distribution. The crosslinked microcapsule size and size distribution of mixed emul-sion droplets decreased with the increasing crosslinking time. The menthol release was a diffusion controlwhich depended on the proportion of amino group in chitosan-to-tripolyphosphate molar ratio and cross-linking time. This work also demonstrated that hydrophilicity/hydrophobicity of the continuous phase anddispersion phase controlled SPG membrane emulsification efficiency and quality of the resulting emulsiondroplets.

Keywords: microcapsules, chitosan, menthol, SPG membrane emulsification, mixed emulsion, controlledrelease

Introduction

Menthol is a cyclic terpene alcohol with three asymmetric

carbon atoms. Among the optical isomers, menthol is the

one that occurs most widely in nature and it is endowed

with the peculiar property to be a fragrance and flavour

compound. For this reason, it is widely used as flavouring

for toothpaste, oral hygiene and personal care products

(Galeotti et al., 2002). Menthol is generally available in

the form of crystals or granules with a melting point of

41–43�C. However, its high volatility and whisker growth

are the very important problems concerning its

applications and shelf life. The microencapsulation

method is an accountable and appropriate technique to

solve some problems (Soottitantawat et al., 2005).

Chitosan, derivatized by deacetylation of chitin, has

been used in many applications because of its biocompat-

ibility, biodegradability, non-toxicity and antibacterial

activity (Rinaudo, 2006). Chitosan can form microcapsules

by various methods such as spray drying, emulsification

and followed by solvent evaporation, ionotropic gelation,

coacervation techniques and so on. It can prevent the loss

of volatile flavours, and enhance stability of the flavour core

materials (Soottitantawat et al., 2005). The capsules remain

Address for correspondence: Suda Kiatkamjornwong, Department of Imaging and Printing Technology, Faculty of Science, Chulalongkorn University,Bangkok 10330, Thailand. Tel: þ66-02-218-5587. Fax: þ66-02-218-5587. E-mail: Ksuda@Chula.ac.th

(Received 8 Jul 2012; accepted 4 Dec 2012)http://www.informahealthcare.com/mnc

498

(Received 8 Jul 2012; accepted 4 Dec 2012)http://www.informahealthcare.com/mnc

498

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stable during the release time. However, the size of the

microcapsules prepared by these methods is difficult to

control, and the size distribution is thus very broad (Wei

et al., 2008). The nature of crosslinked chitosan layers

depended on pH of the crosslinking reaction. At high pH,

chitosan precipitated before the crosslinking reaction took

place. If the reaction occurs, the crosslinked chitosan layers

will be very thin. The microcapsule walls crosslinked

through intermolecular and intramolecular ionic bonding

between chitosan and sodium tripolyphosphate (TPP) take

place more effectively at pH less than 7 (Hsieh et al., 2008).

However, Chenite et al. (2001) stated that at pH values

greater than 6.2, it leads systematically to the formation

of a hydrated gel-like precipitate. The size of the aggregates

increases and phase separation occurs at pH greater than

the pKa (�6.5) of the amino group in chitosan (Krajewska,

2004).

The conventional emulsification devices, such as a high-

pressure dispersing, mechanical stirring and rotor–stator

systems, produce rather polydisperse emulsions and con-

sume high energy (Vladisavljevic and Schubert, 2002). A

membrane emulsification system produces emulsions by

permeating a dispersed phase into a continuous phase

through a membrane having a uniform pore diameter.

The membrane emulsification method makes it possible

to produce monodisperse emulsions and consume less

energy (Joscelyne and Tragardh, 2000; Yasuno et al.,

2002). The most commonly used microporous membrane

for the emulsification is the Shirasu Porous Glass (SPG)

membrane. The SPG membrane is made of SiO2–Al2O3,

with a very narrow pore size distribution. It was fabricated

by Nakashima and Shimizu (1986) via a series of sophisti-

cated heat treatments, an induced phase separation cre-

ated a bi-continuous structure of CaO–B2O3 and SiO2–

Al2O3. Commercially, the pore sizes ranging from 0.1 to

18.0 mm are available. The SPG membrane is inherently

hydrophilic, and it is much easier to get the o/w emulsion

than water-in-oil emulsion (w/o) due to the presence of

negatively charged silanol groups on the surface

(Vladisavljevic et al., 2007). Kiatkamjornwong et al. (2009)

had successfully prepared the microcapsules of chitosan

(0.5–40 mm in diameter) by the conventional stirring

method, in which chitosan is the shell of microcapsules

encapsulating the core material of menthol. The size dis-

tribution of the conventional method is rather polydis-

perse, and thus a better technique of porous glass

membrane emulsification can remedy the large size distri-

bution to a narrow size distribution of microcapsules.

In this study, the SPG membrane emulsification method

was applied to prepare the menthol-loaded sodium TPP

crosslinked chitosan microcapsules. The effects of mem-

brane pore size, the amount of menthol loading and TPP

crosslinking time were investigated on the microcapsule

size and size distribution. After many preliminary investi-

gations were carried with the SPG membrane pore size of

5.2mm, the size distribution of the resulting emulsion drop-

lets was rather broad when compared with the dispersion

mixing method. In addition, the chemicals used in this

study deals the hydrophilicity nature in both phases.

Therefore, the primary emulsion by SPG emulsification

mixed with the secondary emulsion by dispersing method

were studied in order to search for the narrower size dis-

tribution with the inherent merit of SPG membrane emul-

sification. Release kinetics of menthol from the

microcapsules at the specified crosslinking time, concen-

tration, and molar ratio of amino group and TPP were

studied.

Materials and methods

Materials

Chitosan (Sea Fresh Chitosan Lab Co., Ltd., Bangkok,

Thailand) with a degree of deacetylation of 95% and a visc-

osity-average molecular weight (Mv) of 100 000g/mol was

used as received. Sodium TPP (Merck, Hohenbrunn,

Germany) was used as a crosslinker. Fully refined light

mineral oil (Hopewell International Co., Ltd., Bangkok,

Thailand), with a density of 0.83 g/cm3 and a viscosity of

158 Saybolts, was used as an oil phase. Light mineral oil is a

hydrophobic liquid and insoluble in water. Its carbon con-

tent ranges from C10 to C28 (Ivanova, 2012), which affects

the viscosity of emulsion. Menthol (Hong Huat Co., Ltd.,

Bangkok, Thailand) with a molecular weight of

156.27 g/mol and slightly water solubility ranges from 420

to 508 mg/l (UNEP, 2003) depending on its isomers was

used as an encapsulated or core material.

Poly(oxyethylene-2-stearyl ether) or Brij 72, a white waxy

solid with Mw of 359 g/mol (Greensville Co., Ltd.,

Thailand) was used as an oil-soluble surfactant. Sodium

dodecyl sulfate (SDS, Merck, Hohenbrunn, Germany) and

cetyl stearyl alcohol (Kao Co., Ltd., Bangkok, Thailand)

were used as a surfactant and a co-surfactant, respectively.

Poly(vinyl alcohol) (PVA-220, Kuraray Co., Ltd., Osaka,

Japan) with 87–89% of hydrolysis degree, having a viscosity

range of 27.0–33.0 mPa s in a 4% aqueous solution at 20�C

by a Brookfield viscometer (JIS K6726, Can-Am

Instruments, Ltd., Ontario, Canada), was used as a

stabilizer in the continuous phase.

Apparatus

A miniature kit for emulsification with an SPG module was

purchased from Kiyomoto Co., Ltd. (Miyazaki, Japan). A

tubular porous glass membrane with the size of 2 cm

length and 1 cm diameter was installed in the module.

The dispersed phase (oil phase) was stored in a Teflon

vessel (20 ml), which was connected to nitrogen gas inlet.

The continuous phase (water phase) containing a mixture

of SDS and PVA in a 250-ml beaker was stirred at 300 rpm

with a magnetic bar to prevent creaming of the droplets.

With an optimum pressure of nitrogen gas, the dispersed

phase can permeate through the uniform pores of the

membrane into the continuous phase to form droplets.

The droplets were then stabilized by the PVA and SDS

dissolved in the continuous phase.

Microencapsulation of menthol 499

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Preparation of microcapsules via SPGemulsification

Effect of the pore size of membrane on size and size

distribution of microcapsules

The SPG membrane with an average pore size of 1.4 or

5.2mm was used for the emulsification. A simple recipe

for the o/w emulsion is shown in Table 1. The SPG mem-

brane was pre-wetted in the aqueous phase. Light mineral

oil was used as a dispersed phase. The continuous phase

containing PVA and SDS was used. The oil phase was per-

meated through the uniform pores of the SPG membrane

by the predetermined pressure of nitrogen gas into the

aqueous phase to form the o/w emulsion droplets.

The predetermined pressure used was slightly above the

critical pressure (Pc), which is a minimum pressure given to

the dispersion phase that causes it to permeate and pass

through the pores of membrane. In this study, the ranges of

the permeation pressures at 48.9–88.8 kPa for the 1.4-mm

pore size, and 14.1–67.0 kPa for the 5.2-mm membrane

pore size were used.

Effect of menthol loading on droplet size and size

distribution of o/w emulsion via SPG emulsification

The o/w emulsion was prepared through the 5.2-mm mem-

brane pore size. The dispersed phase was the mixture of

menthol, 20 g of light mineral oil, 0.5 g of cetyl stearyl alco-

hol and 0.5 g of Brij 72. The continuous phase was the mix-

ture of PVA and SDS only (without chitosan) in deionized

water. The effect of menthol loading on droplet size and

size distribution of o/w emulsion was investigated by vary-

ing the menthol loading from 0, 5 to 10% w/w. The selected

o/w emulsion from 5% w/w called the primary emulsion

was used for mixing with the secondary emulsion by a dis-

persing method.

Preparation of chitosan microcapsules viaconventional dispersing method

Effects of speed, dispersing time and chitosan concentration

The conventional procedures for preparing the o/w emul-

sion of chitosan encapsulated menthol as the emulsion

microcapsules were as follows. The continuous phase com-

prised 50.0 g of deionized water, 0.05 g of SDS, and then 1%

w/v chitosan solution in acetic acid was added. The oil

phase consisting of 5.0 g of mineral oil was mixed using a

high-speed disperser (T 18 basic digital Ultra-Turrax high-

performance disperser, Ika-Werke� GmbH & Co. KG,

Staufen, Germany) at 6000, 10 000, 14 000 and 16 000 rpm

for 30–120 s dispersing time for studying the effects of dis-

persing speed and dispersing time. Effect of the chitosan

concentration on droplet size and size distribution was also

investigated by varying its concentration from 1, 1.5, to

2.0% w/v in acetic acid. The mixture was continuously

stirred at 400 rpm to prevent coagulation. The emulsion

from this method was used as the secondary emulsion for

mixing with the primary emulsion.

Preparation of chitosan microcapsules via the mixing of

both emulsions

The effect of pH on droplet size and size distribution of the

mixed o/w emulsion was investigated as follows: the pri-

mary and secondary emulsions at a ratio of 1:1 w/w were

mixed and stirred gently at 200 rpm for 1 h. The use of 1:1

w/w ratio was based on stability of the mixed emulsion

obtained afterwards. Five grams of 5% w/w of the TPP

crosslinking agent concentration was dropped in the

mixed emulsion and stirred at 400 rpm for the crosslinking

times of 120 min. The pH of the emulsion was adjusted to

3.6, 5, 7 and 8.9 using an acetic acid solution or a sodium

hydroxide solution, and the pH was measured by a digital

pH meter (model 225, Denver instrument, New York).

Menthol loading in dried microcapsulesand controlled release

Effect of crosslinking times

The chitosan microcapsules were prepared via the mixing

of both emulsions as described above. Effect of crosslinking

time on the menthol loading in dried microcapsules and

release profiles was examined by varying crosslinking times

from 30, 60, 90 to 120 min. The TPP crosslinking agent con-

centration was fixed at 5% w/w and pH of 5.

Effect of TPP crosslinking agent concentration

The chitosan microcapsules were prepared by the same

procedure. The effect of TPP crosslinking agent concentra-

tion was carried out by the varied amount from 1, 5, 10 to

15% w/w at pH 5 and 120 min of crosslinking time. TPP

solution was dropped from a burette with a constant rate

Table 1. A simple recipe for the o/w emulsion.

Component Weight (g)

Continuous phase

Water 200

PVA 3.0*, 4.5y

SDS 1.5

Disperse phase

Light mineral oil 20

Notes: TPP was used as a post-adding ingredient for

crosslinking chitosan shell.

Chitosan was added where needed and mentioned

in the text.

*SPG pore size 1.4 mm.ySPG pore size 5.2 mm.

500 R. Nuisin et al.

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into the emulsion. It was kept at room temperature until

120 min was reached.

Effect of the amino group of chitosan-to-TPP molar ratios

The chitosan microcapsules were prepared by the

same procedure. The effect of amino group of chitosan-

to-TPP molar ratios (by mole) was carried out by the

ratios of 2:1, 4:1, 6:1 and 8:1 at pH 5 and 120 min of cross-

linking time.

Characterization of chitosanmicrocapsules

Size distribution of the emulsion microcapsules

The emulsion microcapsules before and after the crosslink-

ing reaction were observed with an optical microscope

(BH2, Olympus Optical Co., Ltd., Tokyo, Japan).

Diameters of approximately 150 droplets were measured

to calculate the average diameter of the microcapsules.

The number average diameter (de) was calculated accord-

ing to Equation (1). The coefficient of variation (CV) was

determined using Equation (2).

�de ¼Xn

i¼1

di=N ð1Þ

%CV ¼Xn

i¼1

di � de

� �N

2 !1=2,de

0@

1A� 100 ð2Þ

where di is the diameter of the emulsion microcapsules, N

is the total number of the microcapsules, measured, and de

is the number-average diameter of the microcapsules

measured.

Morphology of the microcapsules

Morphology of the emulsion microcapsules was observed

using an optical microscopy (model BH2, Olympus Optical

Co., Ltd, Tokyo, Japan).

Controlled release of menthol from the microcapsules

Chitosan microcapsules of 5 ml were transferred to a 15-ml

test tube, and the tube was centrifuged at 3000 rpm for

30 min. The creamy layer was separated from the aqueous

layer, and then transferred to a vacuum oven (Isotemp

285A, Fischer Scientific, Pittsburg, PA) at 30�C and

4.24 kPa for 12 h for evaporating moisture in the microcap-

sules. The microcapsules were placed in an Infrared

Moisture Determination Balance (IMDB). The menthol

loading in dried microcapsules was calculated from

Equation (3) (Hsieh et al., 2006). Release profiles were

calculated in terms of menthol release (%) with time as

shown in Equation (4). All samples were carried out in

triplicate.

Menthol loading in dried microcapsules ¼Wm�W0

Wm� 100

ð3Þ

Release amount ð Þ ¼Wm �Wm tð Þ

Wm �W0

� �� 100 ð4Þ

where Wm is the weight of the dried microcapsules after the

moisture evaporation. W0 is the weight of microcapsules

after menthol had been evaporated at 120�C. Wm(t) is the

weight of microcapsules at 40�C at a given time t (h) in the

Infrared Moisture Balance (IMB, AD-4715, Kracker

Scientific Inc, Albany, NY). The samples were weighed at

time intervals of 1, 3, 5, 7, 12, 24, 48, 60 and 72 h.

Release kinetic studies of menthol from microcapsules

The release profile of menthol from chitosan microcapsules

was studied and calculated from Equation (4). The release

data of the microcapsules were analyzed by following the

selected mathematical models: zero-order kinetic, first-

order kinetic, Higuchi equation, which describes drug

release as a diffusion processes based on Fick’s law,

square root time dependence (Higuchi, 1963), Baker–

Lonsdale equation, Hixson–Crowell equation and Ritger–

Peppas equation (Ritger and Peppas, 1987), as shown in

Table 2. The nomenclatures are given as follows: Qt and

Q0 are the amounts of menthol released (g/g) after t (h)

and at t¼ 0, respectively; k0 is zero-order rate constant

expressed in units of concentration/time, k is first-order

rate constant (h�1), kH is Higuchi dissolution constant (g/

g/h)�0.5. Baker–Lonsdale (1974) developed the model from

the Higuchi model for controlled release pattern from a

spherical matrix and described as follows: Ft is the fraction

of oil release at time t (Ft¼Qt/Q1), Qt and Q1 are the

amounts of menthol (oil) release at time t and at infinite

time, respectively. Hixson–Crowell stated that the particle

regular area was proportional to the cubic root of its

volume, where Ft¼ 1� (Qt/Q0). Linear regression was

applied for the calculation of correlation coefficient (R2)

Table 2. Mathematical models for release kinetics understudy.

Model Equations

Zero order Qt¼Q0þ k0t

First order log Qt¼ log Q0þ kt/2.303

Higuchi Qt¼ kHt1/2

Baker–Lonsdale 3/2[1� (1� Ft)2/3]�Ft¼ kt

Hixson–Crowell (1� Ft)1/3¼ 1� kt

Ritger–Peppas ln Ft¼ ln kþn ln t

Notes: Qt and Q0 are the amounts of menthol released (g/g) after t (release

time) and at t¼ 0, respectively; k0 is zero-order rate constant (concentra-

tion/time); k is first order rate constant (h�1); kH is Higuchi dissolution

constant (g/g/h)�0.5; Ft is the fraction of oil release at time t.

Microencapsulation of menthol 501

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in order to predict the release behaviour of the

microcapsules.

Results and discussion

Preparation of chitosan microcapsules via SPG

emulsification

Effect of the pore size of membrane on emulsion droplet size

and size distribution

The SPG membranes with the average pore diameters of 1.4

or 5.2 mm were used to prepare the o/w emulsion. The uni-

form emulsion droplets were obtained, and the average

diameter (de) of 10.2 mm with a CV of 10.1 and 19.6 mm

with a CV of 11.5 were produced from the membrane

pore sizes of 1.4 and 5.2mm, respectively. It was found

that the diameter of droplets in the o/w emulsion was

approximately eight and four times the diameters of the

respective membrane pore sizes. The dispersed phase con-

taining light mineral oil without menthol as an additive was

difficult to permeate through the SPG membrane pore size

of 1.4mm. The possible cause was arisen from the high vis-

cosity of light mineral oil (158 Saybolts). Furthermore, the

high hydrolysis degree (87–89%) of PVA stabilizer made the

SPG membrane too hydrophilic. In addition, PVA mole-

cules covered onto the membrane surface to make a smal-

ler pore radius because the PVA molecules possess a

surface tension of 43 mN/m, which is lower than the high

energy of the membrane surface. Furthermore, the wetting

surface of SPG membrane may yield broader size disper-

sion when more hydrophilic oil phase were involved (Omi

et al., 1997).

When a viscous fluid flows through a tube of fixed length

and inner diameter, a resistance to fluid flow exists. The

resistance to fluid flow for steady flow through a circular

tube of radius can be explained by Poiseuille’s law for resis-

tance. Due to friction force induced by the capillary and the

drop of pressure difference, the smaller radius of the mem-

brane can produce the higher resistance to flow as the vis-

cosity increased and thus a small amount of fluid can flow.

As a result, the oil phase thus could not be permeated from

the small pore radius of the membrane, and the larger pore

size membrane of 5.2mm was thus used for future

investigation.

Effect of menthol loading on droplet size and size distribution

The menthol dissolved in the dispersed phase affected the

o/w emulsion. The permeation pressure decreased steadily

from 7.4 to 2.5 and 0.6 kPa when increasing the amount of

menthol from 0% to 5% and 10% w/w, respectively. The

hydrophilicity in the disperse phase increased with an

increasing amount of menthol loading in the disperse

phase. The hydroxyl group of menthol enhances hydrophi-

licity of the oil phase, thus allowing it to permeate easily

through the hydrophilic wall of the SPG membrane. The

permeation pressure was thus decreased. The optical

micrographs and the histogram of size distribution of the

droplets in the oil phase at different amounts of menthol

loadings are shown in Figure 1(a–c). Without the menthol

loading (Figure 1a), an average diameter of the emulsion

droplets of 28.3 mm with a CV of 24.7% was obtained. When

the amounts of menthol loading in the disperse phase

increased from 5 to 10% w/w, the average diameter of emul-

sion droplets decreased from 24.2 to 16.8 mm with the CV of

the droplets increased from 34.4% to 57.6%. The size distri-

butions expressed as the histograms in Figure 1(d–f) indi-

cate that more menthol loading in the dispersed phase

produced the smaller size droplets in the range of 10mm.

Likewise, increases in microcapsule sizes were caused by

the increases in hydrophilicity of the hydroxyl groups when

increasing the menthol loadings.

Normally, fairly uniform (o/w) emulsion droplets with

the CV around 10% are obtained from the SPG emulsifica-

tion (Ma et al., 2004). Then, one may observe the poor

performance of the SPG membrane emulsification against

the high-speed dispersion with respect to the narrower size

distribution because the CVs were relatively greater than

the high-speed dispersion. This poor SPG performance was

caused by the hydrophilic ingredients in the disperse phase

which contained besides menthol, cetyl-steary alcohol and

Brij 72 could produce some hydrophilicity to the disperse

phase and gave the large CV of the size distribution. Thus,

hydrophilicity of the added ingredients in the dispersed

phase closely affects the size distribution of the droplets

formed with SPG membrane.

Preparation of microcapsules via conventional dispersing

method

The o/w emulsion with narrow size distribution by SPG

emulsification technique requires that the emulsion must

be stable throughout the emulsification. Since the oil phase

composed of different amounts of menthol encapsulated

by chitosan, a hydrophilic/hydrophobic nature between

the interphase was required. Therefore, it is necessary to

find a condition where a large amount of chitosan can be

maintained to form a shell on the surface of each droplet to

become microcapsule. The high-performance disperser

was used with a suitable dispersing rate. The effects of dis-

persing speed, time, and chitosan concentration were stud-

ied for this purpose.

Effect of dispersing speed

Chitosan microcapsules with a broad coefficient of varia-

tion of 55.9% were obtained using the lower dispersing

speed of 6000 rpm. It is anticipated that the higher viscosity

of chitosan at the low dispersing speed caused uneven dis-

tribution of the droplets during the droplet formation.

Increasing the dispersing speeds from 14 000 to

16 000 rpm, the microcapsules with CV of 38.9 and 39.7%,

respectively, were obtained. However, the dispersing speed

at 10 000 rpm was found to give the lowest CV of 31.5%, the

emulsion microcapsule droplets were in the size of

39.1� 12.3 mm. Unfortunately, phase separation of the

emulsion was observed during the emulsification process

at approximately 1 h after the dispersion. As shown in

Table 3, the dispersing speed of 14 000 rpm was selected

for further study since the emulsion microcapsule droplets

were in the size of 20.2� 7.9 mm.

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Effect of dispersing time

Chitosan microcapsules were prepared and used as a sec-

ondary emulsion (with chitosan) for combining with the

primary emulsion (without chitosan) already obtained via

the SPG emulsification process. As mentioned above, the

dispersing speed of the disperser was used at 14 000 rpm.

The effects of dispersing time of 30, 60, 90 and 120 s were

then studied. As shown in Table 3, the emulsion droplets

with the CV of 30.8% and 34.8% were obtained with the

dispersing times of 30 and 60 s, respectively. The broad

droplets size distribution was compared with those

obtained with the dispersing times of 60 and 120 s. The

coefficients of variation of 24.8% and 24.6% were obtained.

The shorter the dispersing time is, the greater the droplet

disruption becomes, and the small number of oil droplets

was stabilized by the surfactant. The oil droplet distribution

was related to the stirring time that affected the stability of

the oil droplets in the aqueous phase (Kiatkamjornwong

et al., 2009). With an increasing stirring time from 90 to

120 s, the emulsion droplets size was increased from

39.8� 9.9 to 41.2� 10.1 mm, because more turbulent flows

induced higher diffusion of oil to the existing droplets,

resulting in its larger size. The stirring time of 90 s was

then selected for further studies to find a condition for

the smaller droplets formation.

Effect of chitosan concentration

Chitosan can dissolve better in an acidic solution at pH not

higher than 3. As shown in Table 4, the amount of chitosan

Figure 1. Optical micrographs (a–c) and histograms of the size distribution (d–f) of the o/w emulsion with different amounts of menthol loadings: (a) and

(d) without menthol, (b) and (e) 5 wt% menthol, and (c) and (f) 10 wt% menthol.

Table 3. Effects of speed and dispersion time on size and size distribu-

tion of emulsion.

Run no.* Speed

(rpm)

Dispersion

time

(s)

de � SD

(mm)

CV

(%)

D001 6 000 60 62.6� 34.9 55.9

D002 10 000 39.1� 12.3 31.5

D003 14 000 20.2� 7.9 38.9

D004 16 000 30.5� 12.1 39.7

T30 14 000 30 38.7� 11.9 30.8

T60 60 38.7� 13.5 34.8

T90 90 39.8� 9.9 24.8

T120 120 41.2� 10.1 24.6

Note: *Chitosan solution 1% w/v of 2 M acetic acid, the amount of

chitosan was 10.0 g.

Microencapsulation of menthol 503

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was varied from 1%, 1.5% and 2.0% w/v of acetic acid and

the emulsion droplets of chitosan were in the sizes of

17.9� 8.1, 17.0� 6.3 and 16.7� 6.9mm, with CVs of

45.6%, 37.1% and 41.0%, respectively. The chitosan solution

at 1.5% w/v in acetic acid was considered a suitable con-

centration for the experiments because it gave the smaller

droplets and stable viscosity. The stable viscosity is a major

controlling parameter for the system to emulsify, to perme-

ate from the membrane pores, to form droplets and to form

the outer shell of the microcapsule (Wang et al., 2005).

Preparation of chitosan encapsulated menthol microcap-

sules via mixed emulsion method

One important point for using the mixed emulsion method

instead of using one single step of SPG emulsification pro-

cess is discussed later. The ‘‘mixed’’ emulsification process

was selected rather than the single stage SPG process as

mentioned earlier because the gradients in our system were

not suitable for the single stage SPG process. It is found that

when the continuous phase was composed of PVA, SDS

and chitosan (in acetic acid solution) and mixed together

at pH 4.35, the continuous phase was found with precipi-

tated agglomerates. A single addition of acidic solution of

chitosan into either SDS or PVA solution was performed. In

the continuous phase only SDS and chitosan (in acetic acid

solution) were mixed together at pH 4.32, this phase was

found with precipitated agglomerates of SDS but the PVA

solution with the addition of chitosan acidic solution at the

same pH did not induce any precipitate. This formulation

induces the limitation of using a single SPG emulsification

process for preparing the emulsion in this research. The pH

effect of chitosan solution from pH 3.47 to 4.3 was that

chitosan slightly precipitated in the form of very small gran-

ular-like gel when pH was rose to 4.35.

In details, pH of the primary emulsion was 4.95 and that

of the secondary emulsion (with chitosan) was 3.98. When

mixing the primary emulsion with the secondary emulsion,

the pH of the mixed emulsion was 4.35. Through this mixed

emulsion process, the chitosan-encapsulated menthol

microdroplets were achieved. This is the main reason

that the ‘‘mixed’’ emulsification process was used rather

than the single SPG emulsification process.

Menthol encapsulated microcapsules were thus pre-

pared and stabilized via a mixed emulsion method (o/w)

by mixing two sets of o/w emulsions together. The emul-

sions from the SPG emulsification using the SPG

membrane pore size of 5.2 mm as the primary emulsion

(o/w) can be mixed with another emulsion prepared sep-

arately by the high-speed disperser at 14 000 rpm for 90 s as

the secondary emulsion (o/w) to give the mixed o/w emul-

sion. The miscibility of the two emulsions was studied in

terms of droplet size and encapsulation amount. In this

experiment, the primary emulsion consisting of emulsion

droplets of 24.2� 8.3mm with a CV of 34.4% was mildly

stirred with the secondary emulsion having the droplet

size of 17.0� 6.3mm with a CV of 37.1%. The emulsion

needed an appropriate time to equilibrate and stabilize.

The final emulsion droplets in the size of 31.0� 8.5 mm

with the narrow CV of 27.4% were obtained as shown in

Table 5. It was found that the smaller sizes of the secondary

emulsion droplets diffused and absorbed onto the primary

emulsion droplets until the swollen droplets reached sta-

bility with a narrower size distribution. The type of mech-

anism is the so-called diffusion process (Higuchi and

Misra, 1962; Ma et al., 1997). The diffusion process which

states that the solubility of the substance in the form of

droplets is expressed in the following equation:

C0 ¼ C� exp 2�Vm=rRTð Þ ð5Þ

where C0 is the solubility of the substance in the form of

droplets with radius r, C* is the solubility of the substance

in bulk state, � is the interfacial tension, Vm is the molar

volume of the substance, R is the gas constant and T is the

temperature (K).

According to Higuchi and Misra (1962) and Ma et al.

(1997), the process involves the diffusion of the materials

in the form of droplets in the secondary emulsion into the

aqueous phase and absorbed then by the primary emul-

sion. That is, chitosan (in the secondary emulsion), a more

hydrophilic material, can dissolve and diffuse in the aque-

ous phase and then is absorbed into the higher hydropho-

bic primary emulsion to give the swollen droplets as shown

in Figure 2 and in the following expression:

o=wð Þpþ o=wð Þs! o=wð Þswollen p

where (o/w)p denotes for the o/w primary emulsion, (o/w)s

for the o/w secondary emulsion and (o/w)swollen p for the

final product of the swollen primary emulsion. In addition,

the droplet sizes in both optical micrographs implied some

more observations as follows. The swelling process may

take place in both directions, i.e. the first direction is a

disappearance of the smaller droplets in the secondary

emulsion according to a slight increase of the solubility in

water as mentioned earlier. The second direction is the

diffusion of menthol and Brij 72 in the primary emulsion

Table 4. Effect of chitosan concentration on size and size distribution of

the microcapsules.

Run no. Chitosan

concentration

(% w/v)

de (mm)� SD CV (%)

CH1 1.0 17.9� 8.1 45.6

CH1.5 1.5 17.0� 6.3 37.1

CH2 2.0 16.7� 6.9 41.0

Table 5. The emulsion types, microdroplets size and size distribution.

Conditions de(mm) �SD CV (%)

Primary emulsion 24.2� 8.3 34.4

Secondary emulsion 17.0� 6.3 37.1

Mixed emulsion 31.0� 8.5 27.4

Notes: Primary emulsion using SPG pore size of 5.2 mm.

Secondary emulsion using a dispersing speed at 14 000 rpm for 90 s.

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towards the larger droplets in the secondary emulsion.

When one considers the mixed emulsion, the droplet

sizes look somewhat as large as those in the secondary

emulsion.

Effect of pH on size and size distribution of microcapsules

The effects of pH adjusting solution on the size and size

distribution of the swollen microcapsules were investi-

gated. The pH in the ranges of 5–7 was preferred for the

use in the microcapsules as an additive in leave-on hair

conditioners. However, the effect of pHs of 3.6, 5.0, 7.0

and 8.9 was investigated. As shown in Figure 3, droplet

sizes of the microcapsules tended to decrease to give an

increasing coefficient of variation (26.5%) at pH 3.6. The

microcapsule degradation could result from the non-cross-

linked chitosan molecules and then be dissolved from the

microcapsules at this pH. The pHs of 5.0, 7.0 or 8.9 of the

emulsions were adjusted using the sodium hydroxide solu-

tion. The chitosan-encapsulated menthol microcapsules in

the sizes of 26.8, 28.6 and 28.0 mm were obtained at pHs 5, 7

and 8.9, respectively. The coefficients of variation at the

above-mentioned pHs were 20.6%, 23.7% and 30%, respec-

tively. Then, the workable pH ranges from 5 to 7 were

selected for skin care applications because its pH is in a

neutral range which is suitable for the type of products, and

the microcapsule walls were crosslinked effectively through

intermolecular and intramolecular ionic bonding between

chitosan and TPP.

Menthol loading in dried microcapsules

Effects of crosslinking time

Chitosan-encapsulated menthol microcapsules prepared

via the mixed emulsion technique were crosslinked with

5% w/w TPP under the crosslinking times of 30, 60, 90 or

120 min. The crosslinked chitosan molecules were found to

distribute in both the oil–water interface and in the solution

because the accumulation of chitosan was observed in both

phases. The small amount of the crosslinked compounds

from free chitosan molecules with TPP were also observed

in the bulk solution.

The menthol loading in dried microcapsules are pre-

sented in Figure 4(a). The result revealed that the menthol

was encapsulated by the chitosan shell and increased with

increasing crosslinking time from 30 to 60 min. The men-

thol loading in dried microcapsules became constant at 90

and 120 min. The encapsulation pattern indicated that the

menthol could be released through the controlled thick-

ness of the microcapsule shells. The ionic interaction

between a negatively charged counterion of TPP and a pos-

itively charged amino group of chitosan was presented as

an important role for the degree of crosslinking reaction

(Ko et al., 2002). The menthol loading in dried microcap-

sules remained constant when a longer crosslinking time

was applied to the system. The release of menthol was thus

very slow which could be caused by the thickness of chit-

osan shell. Figure 4(b) shows the release behaviour of men-

thol from the chitosan microcapsules crosslinked by the

TPP solution by the ionic crosslinking agent at various

crosslinking times. Regardless of any crosslinking time,

the release amount (%) increased rapidly with a steep

slope from the beginning of the release to about 10 h of

releasing time to give 60% release. The release decreased

from 45% to 15% when the crosslinking time was from 60 to

120 min. After 10 h of release, the releasing curve increased

slowly to reach asymptotically a plateau value. This implies

that the microcapsule shell thickness controlled the extent

of menthol release which was released constantly at a

longer releasing time. In another words, one can

Figure 2. Optical micrographs illustrating the mixing of primary emulsion and secondary emulsion to give a mixed emulsion.

Figure 3. Effect of TPP crosslinking time at various pHs on average par-

ticle size of the microcapsules.

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manipulate the amount of releasing menthol through the

crosslinking time of chitosan.

Effects of crosslinking concentrations

Figure 5 shows the release behaviour of menthol from the

crosslinked chitosan microcapsules with various concen-

trations of TPP solution. As mentioned previously, the

amount of crosslinking agent influents the ionic-crosslink-

ing density of TPP-chitosan. The menthol loading in the

dried microcapsules in Figure 5(a) was improved by

increasing amounts of TPP concentrations from 1%, 5%,

10% to 15% w/w, to give the menthol loading in the dried

microcapsules at 14.8� 7.8, 36.1� 0.3, 38.1� 1.7 and

37.7� 3.3%, respectively, with a maximum slope

(36.1� 0.3) at 5% w/w TPP concentration. Further

increases in TPP concentrations did not produce any sig-

nificant increases in the menthol loading in dried micro-

capsules. Figure 5(b) shows the effect of the TPP

crosslinking agent on the release behaviour of menthol.

The TPP-crosslinked chitosan microcapsules at 1% w/w

TPP could release 95% of menthol within 60 h. The

amounts of menthol release from the microcapsules were

46.1� 1.5, 43.8� 2.3 and 38.3� 2.5% when TPP concentra-

tions at 5%, 10% and 15% w/w, respectively, were used to

crosslink the chitosan shells. Remunan-Lopez and

Bodmeier (1997) reported that the diffusion of drug from

chitosan films decreased as the concentration of the TPP

solution increased. The high crosslinking density of TPP-

to-chitosan matrix resulted in the formation of a

denser network in the chitosan shell, hence the release of

menthol decreased. Thus, the amount of menthol released

from the microcapsules depends greatly on the thickness of

TPP-crosslinked chitosan shell, i.e. the thicker the shell, the

lower the amount of menthol released.

Effect of concentration of amino group in chitosan-to-TPP

molar ratio

The menthol loading in dried microcapsules increases with

increasing molar ratios of amino group in chitosan to TPP

of 2:1, 4:1, 6:1, and 8:1, as shown in Figure 6(a). Chitosan

microcapsules provided the menthol loading in the dried

microcapsules of 47.5� 3.5%, 52.7� 0.5, 67.0� 1.5% and

69.2� 1.3%, respectively. The releasing profile of menthol

from the crosslinked chitosan microcapsules in Figure 6(b)

were decreased with increasing amounts of the molar ratio

of amino group to TPP. The menthol release was reduced

after 12 h. The amounts of menthol release of 34.0� 2.0%,

22.9� 2.9%, 18.4� 2.9% and 16.2� 2.8% were obtained

when the amino group-to-TPP molar ratios of 2:1, 4:1, 6:1

and 8:1 were used, respectively, at 12 h releasing time. The

slope of releasing profiles for all molar ratios of amino and

TPP increased at the initial stage and relatively constant

after 60 h. Of course, the release rate of menthol was slow

down when the molar ratio of amino and TPP increased.

The chitosan shells became almost solid when the cross-

linked degree was high enough. As mentioned previously in

the literature (Lopez-Leon et al., 2005; Liu and Gao, 2009),

the amino group in chitosan interacts with the TPP disso-

ciated ions of OH�, HP3O4�10 and P3O5�

10 (Ko et al., 2002) to

give crosslinking sites via ionotropic crosslinking or

Figure 4. Dependence of the dried microcapsules properties on TPP crosslinking time and release time: (a) menthol loading of the dried microcapsules

versus TPP crosslinking time and (b) release efficiency versus release time of the dried microcapsules at various TPP crosslinking times.

Figure 5. Dependence of the dried microcapsules properties on TPP concentration: (a) menthol loading of the dried microcapsules versus TPP concen-

tration and (b) release efficiency versus release time of the dried microcapsules at various TPP crosslinking amounts.

506 R. Nuisin et al.

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interpolymer complex. The higher the amount of amino

group, the greater the crosslinking density of the microcap-

sules, and the thicker the microcapsule shells of chitosan

were obtained. Due to the ionic interaction between TPP

and chitosan, TPP crosslinked chitosan aggregates could be

found at two sites in the emulsion. First, the aggregates

formed at the interfaces of the microdroplets shells of chit-

osan, and second, the aggregates dispersed in the aqueous

phase in the form of TPP-crosslinked chitosan debris as by-

products of the emulsion system. The extent of crosslinking

density produced by 2:1 versus 8:1 ratios indicated that

crosslinking density controls significantly the release

amount (%), which was found more profoundly at the

longer releasing time.

Release kinetics studies of menthol from the microcapsules

The release kinetics of the chitosan-encapsulated menthol

from various crosslinking times, crosslink concentrations

and molar ratios of amino to TPP were compared by their

respective correlation coefficients. The mentioned vari-

ables affect sharply only the slope of the initial bursting

period, after 10 h of the release, all the curves proceeded

with roughly the same rate. It could be stated that if the

initial release is preferred for the controlled release of men-

thol, the crosslinking time is the decisive factor. Then, some

specific application in relation between the required

amount released and time can be tailored made.

As mentioned in Table 2, various models and equations

of zero order, first order, Higuchi, Hixson–Crowell, Baker–

Lonsdale, and Ritger–Peppas (Ritger and Peppas, 1987; Shi

et al., 2010) were used to linearize and predict the mecha-

nisms of menthol release. The best model was selected on

the basis of highest value of regression coefficient (R2).

Ritger–Peppas plots were found to give the highest linearity

with the correlation coefficient greater than those of the

other kinetics, as shown in Figure 7(a–c). The Ritger–

Peppas model includes an empirical observation of the ini-

tial burst, as shown in the slope of the curves at the specific

conditions in Figure 7 (crosslinking time, TPP concentra-

tion, and the amino group-to-TPP molar ratio). The results

also indicated that the menthol release mechanism from

the chitosan microcapsules was the diffusion control with a

diffusion index (n) calculated from the slopes. The n values,

in Table 6, reveal that the releasing menthol was a spherical

microcapsule in shape. The diffusion index n was 0.43,

Figure 7. Linear regression of Ritger–Peppas models for release kinetics

of menthol under the effect of (a) crosslinking time, (b) TPP concentra-

tion, and (c) amino group-to-TPP molar ratio.

Figure 6. Dependence of the dried microcapsules properties on amino group-to-TPP and release time: (a) menthol loading of the dried microcapsules

versus the amino group-to-TPP ratios and (b) release efficiency versus release time of the dried microcapsules at various amino group-to-TPP ratios.

Microencapsulation of menthol 507

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which indicates the Fickian diffusion, while 0.43�n� 1.00

is the non-Fickian diffusion (Ritger and Peppas, 1987).

Except a few cases of crosslinking times from 30 to

90 min with the low concentration of TPP at 1%, the rest

of ‘n’ values in the Ritger–Peppas model were approaching

0.5, which implied that the menthol release obeys the non-

Fickian transport and the system that worked under stress

or structural changes and relaxation induced by the visco-

elastic effect of polymer affected the diffusion and con-

trolled release processes. The result demonstrated that

the transportation of menthol was definitely controlled by

the crosslink density (Kulkarni et al., 2011). When the

crosslinking density increased, the value of n also

increased.

Conclusion

Menthol as a volatile core material was encapsulated by

using both the high-speed dispersion and SPG membrane

emulsification techniques having chitosan as an encapsu-

lating shell. The SPG membrane pore size, the amount of

menthol loadings and crosslinking times affected the emul-

sion droplet size and size distribution and release property.

The nearly uniform-sized chitosan encapsulated menthol

microcapsules were achieved when the membrane pore

size used was 5.2mm. The higher amount of menthol

gave the smaller average diameter of the emulsion droplets

with a narrow size distribution. By the longer crosslinking

time, the smaller microcapsules with a narrow size distri-

bution were produced. The emulsion droplets with the

average size of 31.0� 8.5 mm with a coefficient of variation

of 27.4% were obtained from mixing the primary emulsion

by the SPG method and the secondary emulsion by dispers-

ing method. The suitable pH to stabilize the microcapsules

was over the range of 5–7. The menthol loading and release

properties of the dried microcapsules depended on the

amount of TPP crosslinking agent and crosslinking time,

and molar ratio of amino group in chitosan to TPP. The

menthol release was an exponential curve, normally with a

higher rate at the initial portion. The non-Fickian diffusion

was also found in the menthol release from the crosslinked

chitosan microcapsules which depended greatly on the

crosslinking thickness of the chitosan shell. The effective-

ness of SPG emulsification in producing a very narrow size

distribution of emulsion droplets or particles, i.e. small

standard deviation and coefficient of variation, depends

greatly on the hydrophilicity/hydrophobicity of the compo-

nents used in both phases.

Acknowledgements

Many thanks go to the Department of Imaging and Printing

Technology, and the Department of Environmental

Science, both of the Faculty of Science, Chulalongkorn

University for providing research facilities.

Declaration of interest

The authors report no conflicts of interest. The authors

alone are responsible for the content and writing of the

article.

The authors are grateful to the Thailand Research Fund

for providing the research fund under the Senior Scholarly

Consolidation Grant no. RTA5080004.

References

Baker RW, Lonsdale HS. 1974. Controlled release of biologically activeagents. New York: Plenum Press.

Chenite A, Buschmann M, Wang D, Chaput C, Kandani N. Rheologicalcharacterization of thermogelling chitosan/glycerol-phosphate solu-tions. Carbohydr Polym, 2001;46:39–47.

Galeotti N, Di Cesare Mannelli L, Mazzanti G, Bartolini A, Ghelardini C.Menthol: A natural analgesic compound. Neurosci Lett, 2002;322:145–8.

Higuchi T. Mechanism of sustained-action medication: Theoritical analysisof rate of release of solid drugs disperse in solid matrices. J Pharm Sci,1963;52:1145–9.

Higuchi WI, Misra J. Physical degradation of emulsion via the moleculardiffusion route and the possible prevention thereof. J Pharma Sci,1962;51:459–66.

Hsieh FM, Huang C, Lin TF, Chen YM, Lin JC. Study of sodium tripolypho-sphate-crosslinked chitosan beads entrapped with Pseudomonas putidafor phenol degradation. Process Biochem, 2008;43:83–92.

Hsieh WC, Changm CP, Gao YL. Controlled release properties of chitosanencapsulated volatile citronella oil microcapsules by thermal treatments.Colloids Surf B Biointerfaces, 2006;53:209–14.

Ivanova K. Composition, 2012, WIPO Patent Application WO/2012/022516,February 23, 2012.

Joscelyne SM, Tragardh G. Membrane emulsification – A literature review.J Membr Sci, 2000;169:107–17.

Kiatkamjornwong S, Phansomboon S, Hoven V. Chitosan microencapsu-lation of menthol and its controlled release. J R Inst Thai,2009;34:337–71.

Ko JA, Park HJ, Hwang SJ, Park JB, Lee JS. Preparation and characterizationof chitosan microparticles intended for controlled drug delivery. Inter JPharm, 2002;249:165–74.

Krajewska B. Application of chitin- and chitosan-based materials forenzyme immobilizations: A review. Enzyme Microb Tech,2004;35:126–39.

Kulkarni RV, Mangond BS, Mutalik S, Sa B. Interpenetrating polymer net-work microcapsules of gellan gum and egg albumin entrapped withdiltiazem–resin complex for controlled release application. CarbohydrPolym, 2011;83:1001–7.

Liu H, Gao C. Preparation and properties of ionically cross-linked chitosannanoparticles. Polym Adv Technol, 2009;20:613–19.

Table 6. Release rate constant (k), diffusion index (n), and correlation

coefficient (R2) following linear regression of log fraction of menthol

versus log time of the Ritger–Peppas model.

Condition k R2 n

Crosslinking time (min)

30 �1.5433 0.9666 0.4020

60 �2.0190 0.9185 0.4283

90 �2.3721 0.9805 0.3990

120 �2.8679 0.9964 0.5026

Concentration of TPP (%)

1 �1.6865 0.9440 0.4007

5 �2.8671 0.9963 0.5026

10 �2.9490 0.9755 0.5031

15 �2.9940 0.9930 0.4748

Molar ratio of CS:TPP

2:1 �2.6051 0.9398 0.4809

4:1 �2.9606 0.9484 0.4913

6:1 �3.2770 0.9030 0.4848

8:1 �3.3382 0.8972 0.4874

508 R. Nuisin et al.

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nal o

f M

icro

enca

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atio

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ownl

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om in

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ahea

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re.c

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arol

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stitu

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Lopez-Leon T, Carvalho ELS, Seijo B, Ortega-Vinuesa JL, Bastos-Gonzalez D. Physicochemical characterization of chitosan nanoparti-cles: Electrokinetic and stability behavior. J Colloid Inter Sci,2005;283:344–51.

Omi S, Kaneko K, Nakayama A, Katami K, Taguchi T, Iso M,Nagai M, Ma GH. Application of porous microspheres preparedby SPG (Shirasu Porous Glass) emulsification as immobilizingcarriers of glucoamylase (GluA). J Appl Polym Sci, 1997;65:2655–64.

Ma GH, Nagai M, Omi S. Synthesis of uniform microspheres with highercontent of 2-hydroxyethyl methacrylate by employing SPG emulsifica-tion technique followed by swelling process of droplets. J Appl Polym Sci,1997;66:1325–41.

Ma GH, Sone H, Omi S. Preparation of uniform-sized polystyrene-polya-crylamide composite microspheres from a W/O/W emulsion by mem-brane emulsification technique and subsequent suspensionpolymerization. Macromolecules, 2004;37:2954–64.

Nakashima T, Shimizu M. Porous glass from calcium alumino boro-silicateglass. Ceramics, 1986;21:408.

Remunan-Lopez C, Bodmeier R. Mechanical, water uptake and permeabil-ity properties of crosslinked chitosan glutamate and alginate films.J Control Release, 1997;44:215–25.

Rinaudo M. Chitin and chitosan: Properties and applications. Prog PolymSci, 2006;31:603–32.

Ritger PL, Peppas NA. A simple equation for description of solute release I.Fickian and non-Fickian release from non-swellable devices in the formof slabs, spheres, cylinders or discs. J Control Release, 1987;5:23–36.

Shi K, Jiang Y, Zhang M, Wang Y, Cui F. Tocopheryl succinate-based lipidnanospheres for paclitaxel delivery: Preparation, characters, and in vitrorelease kinetics. Drug Deliv, 2010;17(1):1–10.

Soottitantawat A, Takayama K, Okamura K, Muranaka D, Yoshii H,Furuta T, Ohkawara M, Linko P. Microencapsulation of l-menthol byspray drying and its release characteristics. Innovat Food Sci EmergTech, 2005;6:163–70.

UNEP Publications, Menthols, SIDS Initial Assessment Report for SIAM 16,Paris, 27-May 2003, p. 8.

Vladisavljevic GT, Kobayashi I, Nakajima M, Williams RA, Shimizu M,Nakashima T. Shirasu Porous Glass membrane emulsification:Characterization of membrane structure by high-resolution X-ray micro-tomography and microscopic observation of droplet formation in realtime. J Membr Sci, 2007;302:243–53.

Vladisavljevic GT, Schubert H. Preparation and analysis of oil-in-wateremulsions with a narrow droplet size distribution using Shirasu-porous-glass (SPG) membranes. Desalination, 2002;144:167–72.

Wang LY, Ma GH, Su ZG. Preparation of uniform sized chitosan micro-spheres by membrane emulsification technique and application as acarrier of protein drug. J Control Release, 2005;106:62–75.

Wei W, Wang LY, Yuan L, Yang XD, Su ZG, Ma GH. Bioprocess of uniform-sized crosslinked chitosan microspheres in rats following oral adminis-tration. Eur J Pharm Biopharm, 2008;69:878–86.

Yasuno M, Nakajima M, Iwamoto S, Maruyama T, Sugiura S, Kobayashi I,Shono A, Satoh K. Visualization and characterization of SPG membraneemulsification. J Membr Sci, 2002;210:29–37.

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