Food Chemistry 220 (2017) 123–128

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Food Chemistry

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Improving the encapsulation efficiency and sustained release behaviourof chitosan/b-lactoglobulin double-coated microparticles by palmiticacid grafting

http://dx.doi.org/10.1016/j.foodchem.2016.09.1560308-8146/� 2016 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Department of Food Science and Technology, SejongUniversity, 209 Neungdong-ro, Gwangjin-gu, Seoul 05006, Republic of Korea.

E-mail addresses: hjyang@sju.ac.kr (H.-J. Yang), sherrene_lps@yahoo.com.sg(P.S. Lee), choohc2002@naver.com (J. Choe), sjs007kr@hanmail.net (S. Suh),sanghoonko@sejong.ac.kr (S. Ko).

Han-Joo Yang, Pei Sia Lee, Jaehyeog Choe, Seokjin Suh, Sanghoon Ko ⇑Department of Food Science and Technology, Sejong University, Republic of Korea

a r t i c l e i n f o

Article history:Received 11 June 2016Received in revised form 18 August 2016Accepted 26 September 2016Available online 28 September 2016

Keywords:Chitosanb-LactoglobulinPalmitic acidGraftingMicroparticleSustained release

a b s t r a c t

Chitosan (CS) was grafted with 0.1 and 0.5% (w/v) palmitic acid (PA) to improve its encapsulationefficiency (EE) and sustained release characteristics when forming CS microparticles. Thereafter,PA-grafted CS (PA-CS) microparticles were coated with denatured b-lactoglobulin (blg), which formsan outer protective layer. The possibility of hydrophobic interaction with the hydrophobic substancesin the CS microparticles increased as the proportion of the grafted PA increased. EE was measured as64.79, 83.72, and 85.00% for the non-grafted, 0.1, and 0.5% PA-CS microparticles, respectively. In simu-lated small intestinal conditions, 4.66 and 17.55% of the core material release in the PA-CS microparticleswere sustained after 180 min by 0.1, and 0.5% PA grafting, respectively. PA grafting enables the sustainedrelease in simulated gastrointestinal fluids by enhancing the hydrophobic interaction between CS and thehydrophobic core material.

� 2016 Elsevier Ltd. All rights reserved.

1. Introduction Hydrophobic modification of carbohydrate polymers is a pro-

The encapsulation of bioactive compounds in chitosan (CS)/protein double-coated microparticles has been investigated as apotential carrier to achieve site-targeted delivery and prolongedrelease. It is well known that this unique core-shell architecturedesign can withstand the low pH in the stomach owing to theexternal coating layer, composed of an acid-resistant protein, suchas denatured b-lactoglobulin (blg) (Boye & Alli, 2000; Schmidt &Markwijk, 1993). CS microparticles, which appropriately sustainthe diffusion of the encapsulated core material from the shell layer,play an important role in the prolonged release. Unfortunately,most bioactive compounds are weakly or non-charged molecules,which probably results in weak binding with the CS microparticles.Consequently, the stability of the CS microparticles as well as theencapsulation efficiency (EE) might be reduced. In general,hydrophobically modified CS structure is more stable due to itsslow dissociation in aqueous media. Moreover, it provides a highpossibility for the bioactive compounds to be encapsulated in theCS microparticles resulting in an increase in the EE and a moresustained release.

cess in which hydrophobic functional groups, such as alkyl, aralkyl,and deoxycholic acid are added to a water-soluble polymer. Assuch, this hydrophobically modified polymer is able to self-aggregate in water due to the intra- or intermolecular hydrophobicinteractions (Liu, Desai, Chen, & Park, 2005). The glucosamine unitin CS, which is comprised of hydroxyl and amine groups, is themajor functional group through which the hydrophobicity of CScan be modified (Hsieh, Huang, Lin, Chen, & Lin, 2008). In recentstudies, the hydrophobic properties of CS have been successfullyimproved via grafting hydrophobic long-chain fatty acids, such asstearic acid (SA), onto the amino groups of CS (Hu, Ren, Yuan,Du, & Zeng, 2006). However, the long carbon chains of saturatedSA have a relatively high melting point and hardly disintegrate inthe gastrointestinal (GI) tract.

Our research hypothesis is that the structure of CS can bechanged by hydrophobic modification, such as coupling withthe carboxyl groups in palmitic acid (PA), which acts as ahydrophobic chain donator. PA, a hexadecanoic saturated fatty acid(C16H32O2), can be used for grafting the CS molecules. The shorthydrocarbon chain of PA allows the hydrophobically modified CSto disintegrate more easily in the human GI tract. Moreover, PAis stable and easy to handle compared with other unsaturated fattyacids which are susceptible to oxidation. It was assumed that thegrafting reaction with PA could strengthen the emulsifying abilityof CS, which might result in an increase in the encapsulationefficiency. In this study, brilliant blue (BB) was incorporated into

124 H.-J. Yang et al. / Food Chemistry 220 (2017) 123–128

the PA-grafted CS/denatured blg microparticles as a model corematerial. Hence, the outer hydrophilic layer surrounding theacid-soluble CS may protect the internal core material (BB) andallows high stability of the microparticles in the aqueous system.The release behaviour of the core material can be controlled bychanging the grafting ratio. In addition, the additional layer ofdenatured blg can provide resistance against the low pH in thestomach (Das & Kinsella, 1989).

The objective of this study was to develop PA-graftedCS/denatured blg double-coated microparticles to improve the EEand the sustained release behaviour of the core material.

2. Materials and methods

2.1. Materials

CS (22 kDa, degree of deacetylation approximately 84%) waspurchased from Hunan Huasheng Biotech Incorporation(Tianchang, Anhui, China). Powdered blg (98.0% dry basis and93.4% total protein) was donated by Davisco Foods International(Le Sueur, MN, USA). Coomassie brilliant blue (BB) was purchasedfrom Amresco Inc. (Solon, OH, USA). PA (purity 99%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and other analyticalgrade reagents, such as HCl, CH3COOH, NaOH, and NaHCO3, werepurchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Enzymes such as pepsin from porcine gastric mucosa(800–2000 units/mg protein), lipase, pancreatin from porcinepancreas (15–35 units/mg pancreas), and porcine bile extract werealso purchased from Sigma-Aldrich Co. Distilled and deionizedwater used for all solutions in this study were obtained from awater purification system (Aqua MAXTM-Ultra, Younglin InstrumentCo. Ltd., Anyang, Korea).

2.2. Preparation of palmitic acid-grafted chitosan (PA-CS)microparticles

One gram of CS powder was mixed with 10 ml of 1 M NaOHsolution, and then stirred continuously for 2 h at 70 �C (Gan,Wang, Cochrane, & McCarron, 2005). The impurities or insolubleCS were filtered out using a Buchner funnel under vacuum fol-lowed by washing 3 times with distilled water. The purified CSpowder (0.2 g) was then completely dissolved in 20 ml of deion-ized water. PA solutions of different concentrations (0.1, 0.2, 0.3,0.4, and 0.5%) were prepared using ethanol. Then, the CS solutionwas mixed with the different concentrations of PA solutions at areaction ratio of 5:1. After that, 3 ml of EDC (0.7 g/l in distilledwater) was added to the mixture dropwisely. The reaction mixturewas then magnetically stirred at 300 rpm. After 3 h, the finalcoupling reaction was carried out in a shaking water bath at60 �C for 12 h. The reaction mixture was then poured into 200 mlof ethanol/NaOH solution (7/3, v/v) while stirring. The precipitatedmaterial was separated via filtration followed by washing it at least3 times using distilled water and ethanol. Finally, the obtainedmaterial was dried under vacuum condition for 24 h.

2.3. Preparation of BB-loaded PA-grafted chitosan/denatured blg(PA-CS/blg) microparticles

BB solution (0.3 g/l) was prepared by dissolving 0.3 g of BB pow-der in 450 ml of methanol followed by adding 100 ml of acetic acidglacial and 450 ml of distilled water. The BB solution (2 ml), whichacts as a core material, was added to 20 ml of the CS solutionduring the preparation of the PA-CS microparticles. In order to pre-pare PA-CS/blg microparticles, denatured blg solution (0.5%, w/v)was prepared by adding blg powder to distilled water and homog-enizing the mixture using a commercial homogenizer (HG-15A,

Daihan Scientific Co., Ltd., Wonju, Korea) for 15 min. Subsequently,the blg solution was heated at 85 �C under magnetic stirring at800 rpm for 1.5 h followed by ultra-sonication for 20 min. TheBB-loaded PA-CS microparticles (0.2 g) were reconstituted indeionized water (20 ml) and 40 ml of 0.5% (w/v) denatured blgsolution was mixed with them slowly. Thereafter, the pH ofmixture was adjusted to 5.5 using 0.1 M HCl and NaOH followedby magnetic stirring at 500 rpm for 1 h.

2.4. Fourier transform infrared spectroscopy (FT-IR)

The IR spectrum was obtained using FT-IR spectrometer Nicolet380 (Thermo Electron Corp., Waltham, MA, USA) fitted with anattenuated total reflectance mode cell. About 100 ll of the PA-CSmicroparticle suspension was placed in the NaCl plate and sub-jected to light within the infrared region. The instrument was oper-ated at 25 �C, with fixed parameters of resolution, 4 cm�1 and 64times of scans, to obtain the spectrum of each sample. The changesor the intensity of the sample peaks were analyzed via the trans-mittance spectrum in the range of wavelengths 500–4000 cm�1.

2.5. PA-CS coupling efficiency (CE)

The degree of the reaction efficiency or the PA-CS couplingefficiency was determined by the difference in the concentrationof the sample before and after the coupling process, which wasmeasured by using spectrophotometry. The CE was then calculatedusing the following equation:

CEð%Þ ¼ Absorbance of the supernatant after coupling reactionInitial absorbance of sample

� 100

2.6. Transmission electron microscopy (TEM)

The morphology of the samples was examined using TEM(JEM-2010, Jeol Ltd., Tokyo, Japan). The specimens were preparedon Formvar filmed grids and were observed using a transmissionelectron microscope operating at an accelerating voltage of 100 kV.

2.7. Zeta potential measurement

The surface charge of the microparticles was measured using acommercial zeta potential analyzer (Delsa Nano C, Beckman Coul-ter Inc., Fullerton, CA, USA). Each sample was dispersed in distilledwater and continuously scanned 10 times from pH 2 to pH 7 at25 �C. All measurements were carried out in triplicate.

2.8. Encapsulation efficiency (EE)

The encapsulated BB content was measured by a UV-visiblespectrophotometer (Du 730, Beckman Coulter Inc.) at a wavelengthof 500 nm. The EE was determined via centrifugation at 3000 � gfor 8 min to separate the encapsulated and the non-encapsulatedBB. The concentration of BB in the supernatant was measuredand used to calculate the EE.

EEð%Þ ¼ 1�Absorbance of free BB unencapsulated in supernatantAbsorbance of total BB added as core material

� �

�100

2.9. In vitro release behaviour in simulated gastrointestinal fluids

The procedures used to assess the in vitro release behaviourwere slightly modified from a previous study (Garrett, Failla, &

H.-J. Yang et al. / Food Chemistry 220 (2017) 123–128 125

Sarama, 1999; Shim & Kwon, 2010; Wilfart et al., 2008). The BB-loaded microparticle suspension (5 ml) was added to a 100 ml flaskcontaining 25 ml of 20 mM phosphate buffer at pH 6.8. The solu-tion was then mixed gently for about 2 min and its pH wasadjusted to 2.0 using 1 M HCl. For the simulated gastric conditions,1 ml of porcine pepsin (from porcine gastric mucosa, 40 mg/ml in0.1 M HCl) was added. Then, the flask was capped and placed ina shaking water bath (Han Yang Scientific Equipment Co., Ltd,Seoul, Korea) at 150 rpm, 37 �C. Samples were collected at 0, 5,10, 20, 30, 60, 90, and 120 min intervals followed by centrifugationat 3000 � g for 8 min, and BB concentration in the supernatant wasdetermined at each time interval using UV–visible spectrophotom-etry at a wavelength of 500 nm.

Next, the pH of sample was raised to 5.3 using 25 mM NaHCO3

for the simulated small intestinal conditions. Then, 1.5 ml of themulti-enzyme mixture containing 0.2 mg/ml lipase, 2.4 mg/ml bileextract, and 0.4 mg/ml pancreatin in NaHCO3 solution was added.Thereafter, the pH was adjusted to 6.8 using 1 M NaOH. The flaskwas re-capped and placed in the shaking water bath. Samples werecollected at 0, 10, 20, 30, 60, 120, 180, and 240 min intervals fol-lowed by centrifugation at 3000 � g for 8 min. The amount of BBreleased from the microparticles was determined from the pre-post difference in the absorbance during the in vitro release test.

3. Results and discussion

3.1. Physicochemical properties of the PA-CS microparticles

The PA-CS microparticles were prepared in the presence of awater-soluble cross-linker, EDC. The grafting reaction occurred

Fig. 1. (A) TEM images of PA-CS microparticles. (B) FT-IR Spectra

between the carboxyl groups of PA and the amino groups of CS,which resulted in the formation of amide branches and the libera-tion of one molecule of water in equilibrium. In Fig. 1A, the PA-CSmicroparticles prepared using 0.5% (w/v) PA and 1% (w/v) CS areshown, where the darker zone inside the particles was referredto as CS. The diameter of the aggregated particles ranged between1 and 2 lm. Generally, adequate hydrophobic groups and optimumreaction conditions, such as the pH, concentration, and the ratiobetween the two reacting groups are prerequisites to obtainsmall-sized and packed particles.

In this study, a series of PA-CS microparticles were obtained byadjusting the concentration of PA. The formation of microparticlesupon the binding of PA and CS was confirmed by FT-IR spec-troscopy. As shown in Fig. 1B, a structural change in CS after theacylation process demonstrated that PA was successfully graftedonto the chains of CS. In the IR spectra, the peak obtained around3400–3250 cm�1 is referred to as N–H stretching, which corre-sponds to the primary amine functional group in CS (Jiang, Quan,Liao, & Wang, 2006). When PA was grafted, it altered the structureof the CS molecules. The N–H stretching peak in the non-grafted CSmicroparticles (0% PA-CS) was found at 3353.62 cm�1; however, itwas slightly shifted to 3346.05 cm�1 when 0.5% of PA was grafted.An additional peak at 1250–1000 cm�1 was observed, whichpossibly referred to the C-N stretching caused by the presence ofan amide linkage between the amino groups of CS and the carboxylgroups of PA. Hence, no peak was found within this range for thenon-grafted CS microparticles due to the absence of the amidelinkage.

When CS was grafted with PA as listed in Table 1, the CEincreased with increasing PA concentration used for the grafting

of CS microparticle grafted with various concentration of PA.

Table 1Effect of PA concentration on coupling efficiency and surface charge of PA-CSmicroparticles.

Palmitic acidconcentration (PA,%)

Coupling efficiency(%)

Zeta potential(mV)

0 4.26 ± 0.06 38.11 ± 0.860.1 84.89 ± 1.73 27.71 ± 0.080.2 89.37 ± 1.70 28.19 ± 0.450.3 91.36 ± 1.10 28.45 ± 0.470.4 91.88 ± 1.47 29.03 ± 0.680.5 92.03 ± 1.60 30.62 ± 0.51

126 H.-J. Yang et al. / Food Chemistry 220 (2017) 123–128

process, because the hydrophobic substitution of PA increased theavailability (binding ability) of the carboxyl groups. The increase inPA concentration may enhance the chance of hydrophobicinteractions with the CS molecules resulting in the formation ofPA-CS microparticles. Therefore, when PA was added up to anappropriate level, the CE increased. In Table 1, when PA was addedfrom 0.1 to 0.5% (w/v), the CE increased from 84.89% up to 92.03%.

In Table 1, all zeta potential values of the PA-CS microparticleswith 1% (w/v) CS and different concentrations of PA exceeded25 mV, which indicated a moderate stability of the PA-CSmicroparticles in this dispersion system. Zeta potential indicatesthe stability or the tendency of aggregation in a dispersion system.Zeta potential of the PA-CS microparticles was dependent on PAconcentration used in the grafting process and increased asthe concentration of PA increased. This could be attributed to theenhanced hydrophobic interactions between the PA and CS mole-cules at high PA concentrations, which resulted in an increase inthe surface charge of the PA-CS microparticles.

3.2. Physicochemical properties of BB-loaded PA-CS and PA-CS/blgmicroparticles

Fig. 2 shows the effect of PA concentration on the EE of thePA-CS and PA-CS/blg microparticles encapsulating BB as a corematerial. The EE increased with increasing PA concentration (fromnon-grafted up to 0.5%, w/v) in the PA-CS microparticles. The EEwas 64.79, 83.72, and 85.00% for the non-grafted, 0.1, and 0.5%PA-CS microparticles, respectively. There was a slight increase in

Fig. 2. Effects of PA concentration on encapsulation efficiency and zeta potential ofBB loaded PA-CS and PA-CS/blg microparticles.

EE of 0.1 and 0.5% (w/v) PA-CS/blg microparticles compared withthat of the non-grafted ones. The EE was as high as 95.35 and96.83% when 0.1 and 0.5% PA were grafted, respectively.Consequently, the amount of the non-encapsulated BB in thenon-grafted CS microparticles was significantly higher than thatin the PA-CS microparticles, whereas no significant difference inthe amount of non-encapsulated BB was observed between thenon-grafted and PA-CS/blg microparticles. It was considered thatthe hydrophobic PA segments could be easily associated with thehydrophobic core material, BB. Consequently, the increase in PAcontent enhanced the possibility of hydrophobic interactionbetween BB and PA-CS microparticles.

The changes in the zeta potential of the BB-loaded PA-CS andPA-CS/blg microparticles are shown in Fig. 2. The zeta-potentialvalues of the BB-loaded PA-CS/blg microparticles were lower thanthose of the BB-loaded PA-CS microparticles, 21.13 and 22.44 mV,respectively. This occurred because the experiment wasconducted at pH 5.5, which was close to the isoelectric point ofblg (pH 4.7–5.2). Therefore, it might be possible to obtain morestable particles (>± 25 mV) if the pH could be manipulated.Nevertheless, the concentration of PA did not result in any signifi-cant change for both samples.

The surface charge is strongly dependent on the pH and theionic strength of the molecules. CS is acid-soluble and its aminogroups can be protonated as –NH3

+, which may contribute to thepositive charge on the particles (Marguerite, 2006; Pillai, Paul, &Sharma, 2009; Rinaudo, Pavlov, & Desbrieres, 1999). However,when CS was combined with the negatively charged residuesof blg by ionic or electrostatic interactions at pH 5.5, the zeta-potential value was slightly lowered.

3.3. Stability of PA-CS and PA-CS/blg microparticles in simulatedgastrointestinal fluids

Fig. 3A shows the stability of the PA-CS and PA-CS/blg micropar-ticles in simulated gastric fluid. In Fig. 3A, the absorbance of the CSmicroparticles grafted with PA at various concentrations remainedalmost unchanged, which means that no degradation of the PA-CSmicroparticles occurred for 120 min in simulated gastric fluid. InFig. 3B, the results are similar to that in Fig. 3A, which indicatesthat both PA-CS and PA-CS/blg microparticles are stable and canprotect the encapsulated core materials in the gastric environment.The CS microparticles were nearly well dispersed under the strongacidic environment of simulated gastric fluid. For the PA-CSmicroparticles, the acid dispersible characteristic of CS decreasedas the concentration of PA increased. However, the strong acid inthe gastric fluid might continuously hydrolyze the amide bond inPA-CS, which can probably result in the release of the encapsulatedcore material from the microparticles (Agnihotri, Mallikarjuna, &Aminabhavi, 2004). In addition, CS chain formed irregularnetwork-like structure which can enclose the space when the CSmade the particle such as hydrogel bid. Therefore, very tiny gapsor pores are inevitably formed between CS chains. Though the sta-bility of CS microparticles might be able to be increased by makingthe compact CS structures or grafting with other compounds whichcan enhance its hydrophobic characteristics, we cannot completelyprevent the core material from being released by the external fac-tors, such as diffusion, shear force, and chemical degradation(Arifin, Lee, & Wang, 2006). Therefore, an additional outer layercomposed of blg could be suggested as a protective layer for theCS encapsulation system. The resistance of blg against the acidicfluid could help to decrease the hydrolytic degradation of the CSmicroparticles and prevent the encapsulated core material frombeing released in simulated gastric fluid (Fig. 3B) (Bromley,Krebs, & Donald, 2005; Chen, Remondetto, & Subirade, 2006;

Fig. 4. Amount of BB released from (A) PA-CS and (B) PA-CS/blg microparticles insimulated gastric fluid.

Time (min)

0 20 40 60 80 100 120 140

Abso

rban

ce

0.00

0.02

0.04

0.06

0.08

0.10Non-grafted0.1% PA grafted0.5% PA grafted

A

Time (min)

0 20 40 60 80 100 120 140

Abso

rban

ce

0.00

0.02

0.04

0.06

0.08

0.10Non-grafted0.1% PA grafted0.5% PA grafted

B

Fig. 3. Stability of (A) PA-CS and (B) PA-CS/blg microparticles in simulated gastricfluid.

H.-J. Yang et al. / Food Chemistry 220 (2017) 123–128 127

Guzey & McClements, 2006). Thus, through this enhancement, itcan be assumed that the encapsulated core material can be deliv-ered mostly to its target site in GI tract.

3.4. Kinetics of BB release in simulated gastric fluid

Fig. 4 shows the release behaviour of BB in simulated gastricfluid from the PA-CS and PA-CS/blg microparticles, which weregrafted with different concentrations of PA. In simulated gastricfluid, 52.31% of BB was released from the non-grafted CS micropar-ticles after 120 min, whereas it decreased to 45.49 and 31.23% withthe 0.1 and 0.5% (w/v) PA-CS microparticles, respectively. Theincreased concentration of the grafted PA could enhance thepossibility of hydrophobic interaction between BB and the coatmaterials in the particle matrix. Consequently, BB releasedecreased with increasing the concentration of the grafted PA.

In Fig. 4, the PA-CS/blg microparticles showed slower releasepattern than that of the PA-CS microparticles, and the amount ofBB released from the PA-CS/blg microparticles was less than thatreleased from the PA-CS microparticles. The release behaviour ofcore material can be more closely related to the degree ofhydrophobic modification on the CS structure as indicated by PAconcentration and the outer protective layer of blg. The release rateof BB was close to zero as the grafted PA concentration increased inthe PA-CS/blg microparticles; it demonstrated a constant rate or azero-order release of BB over time.

3.5. Kinetics of BB release in simulated small intestinal fluid

Fig. 5 shows the release behaviour of BB in simulated smallintestinal fluid from the PA-CS and PA-CS/blg microparticles, whichwere grafted with different concentrations of PA. As shown inFig. 5, the rate of BB release was directly correlated with theincubation time. BB release was successfully sustained byadditional PA grafting. Approximately 4.66% and 17.55% of BBrelease from the PA-CS microparticles were sustained after180 min via additional 0.1 and 0.5% (w/v) PA grafting, respectively.Hydrophobic modification of CS via grafting with the carboxylgroups of PA was able to prolong the release of BB. The hydropho-bic behaviour of the PA-CS microparticles increased by increasingthe concentration of PA, which consequently prolonged the rateof BB release in the simulated small intestinal fluid.

As shown in Fig. 5, release of BB from the PA-CS/blg micropar-ticles was even more sustained in simulated small intestinal fluiddue to the additional protective layer of blg. BB release decreasedslowly during the initial 2 h and remained constant over the incu-bation time in simulated small intestinal fluid. The initial amountof BB decreased sharply, especially in the non-grafted CS micropar-ticles and complete release of BB occurred after 3 h of incubation insimulated small intestinal fluid. The remaining concentration of BBin the non-grafted CS microparticles was as low as 8.11% comparedto that in the PA-CS microparticles (maximally 25.66% for 0.5% PAgrafting). This means that the increase in PA concentration helped

Fig. 5. Amount of BB remained in (A) PA-CS and (B) PA-CS/blg microparticles insimulated small intestinal fluid.

128 H.-J. Yang et al. / Food Chemistry 220 (2017) 123–128

to prolong the release rate of BB from both PA-CS and PA-CS/blgmicroparticles.

4. Conclusions

The encapsulation efficiency, which relied on the interactionbetween the encapsulated core and coat material, is one of thecrucial factors in the development of delivery systems. In thisrespect, we could enhance the encapsulation efficiency throughhydrophobic modification of the molecular structure of CS by graft-ing the carboxyl groups of PA to form an amide linkage between PAand CS in the presence of a cross-linker, EDC. Therefore, thehydrophobic core materials may be well associated with thePA-CS and PA-CS/blg microparticles, which results in the sustainedrelease characteristics in the simulated gastrointestinal fluids.

Results from the in vitro release study confirmed that incorpo-ration of PA onto CS changed the rate of the core material release;the increase in PA concentration used in the grafting processprolonged the release of the core material. In addition, for site-targeted delivery (such as small intestine or colon), double-layer

coating by adding blg to the PA-CS microparticles as a protectivelayer could overcome the acidic environment of the gastric fluid.

In conclusion, CS is the promising coat material for developingdelivery systems for the bioactive substances, whereas the encap-sulation efficiency could be improved through grafting with fattyacids, such as PA. The novel encapsulation model composed ofPA-CS/blg microparticles might be the most suitable design forthe targeted delivery in the GI tract.

Acknowledgements

This research was supported by Basic Science ResearchProgram through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(No. 2014M3A7B4051125).

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