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International Journal of Pharmaceutics 448 (2013) 19– 27

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics

j o ur nal ho me page: www.elsev ier .com/ locate / i jpharm

ethylated N-(4-N,N-dimethylaminocinnamyl) chitosan-coatedlectrospray OVA-loaded microparticles for oral vaccination

ittaya Suksamrana, Tanasait Ngawhirunpata, Theerasak Rojanarataa,arayuth Sajomsangb, Tasana Pitaksuteepongc, Praneet Opanasopita,∗

Pharmaceutical Development of Green Innovations Group (PDGIG), Faculty of Pharmacy, Silpakorn University, Nakhon Pathom 73000, ThailandNational Nanotechnology Center, Thailand Science Park, Pathumthani, ThailandFaculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand

a r t i c l e i n f o

rticle history:eceived 2 January 2013eceived in revised form 13 February 2013ccepted 4 March 2013vailable online 19 March 2013

eywords:

a b s t r a c t

The purpose of this study was to prepare microparticles entrapping ovalbumin (OVA) as a model anti-gen to induce immune responses in mice following oral vaccination. In this study, calcium-alginate andcalcium-yam-alginate microparticles were prepared by crosslinking alginate with calcium chloride solu-tion using an electrospraying technique. 0.1% (w/v) of methylated N-(4-N,N-dimethylaminocinnamyl)chitosan (TM65CM50CS) was used to coat microparticles entrapping an initial OVA of 20% w/w to poly-mer. The results indicated that the coated microparticles were spherical and had a smooth surface, with

lginatehitosan derivativesicroparticlesral vaccine deliveryvalbumin

an average size of 1–3 �m, and were positively charged. In addition, the particles demonstrated a greaterswelling and mucoadhesive properties than did uncoated microparticles. The in vitro release from themicroparticles indicated that the coated microparticles resulted in more sustained release than uncoatedmicroparticles. The cytotoxicity results showed that all of the formulations were safe. The in vivo oraladministration demonstrated that at the same amount of 250 �g OVA, coated microparticles exhibitedthe highest in vivo adjuvant activity in both IgG and IgA immunogenicity.

. Introduction

Vaccines are biological preparations that improve immunity to aarticular disease. Most vaccines are delivered via parenteral routesGiudice and Campbell, 2006). However, injections are inconve-ient for patients, and injectable formulations normally have highroduction costs. Therefore, mucosal vaccinations are a subjectf great interest due to their simplicity and convenience. More-ver, the oral route stimulates the immune system to produce IgGntibodies in the serum and to generate a mucosal IgA antibodyesponse along the mucosal surfaces of the gastrointestinal (GI)ract (Borges et al., 2005). However, oral vaccination is extremelyifficult due to its extremely low bioavailability. The developmentf oral vaccine formulations requires overcoming numerous obsta-les such as the low permeability of large molecules, lack of drugipophilicity, and inactivation or rapid enzymatic degradation in theI tract. To solve these problems, a considerable number of poly-

eric microparticles are under investigation as delivery vehicles to

he intestine that can protect their cargo from adverse conditionshat could affect vaccine bioactivity (Singh and O’Hagan, 1998).

∗ Corresponding author. Tel.: +66 34 255800; fax: +66 34 250941.E-mail addresses: opraneet@hotmail.com, praneet@su.ac.th (P. Opanasopit).

378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijpharm.2013.03.015

© 2013 Elsevier B.V. All rights reserved.

Starch is one of the most important natural organic com-pounds and is abundant in nature. In recent years, pharmaceuticalcompanies have widely used starches in various stages of drugdevelopment technology. Excipients play a very important role insolid dosage formulations by imparting mechanical strength, sta-bility and tablet disintegration. The yam (Dioscorea sp.) is a localplant in many countries including Nigeria, Jamaica, Brazil, China,and Thailand. Yam starches have played a major role in the foodand pharmaceutical industries over the past few decades (Yu et al.,1999). In Thailand, the most common yam species is Dioscoreaesculenta (Nattapulwat et al., 2009). Yam starches possess the prop-erties of tolerance to shearing and stability in acidic conditions andcan be substituted for modified starches as a functional ingredientfor low pH processed foods. The application of starches from thisyam to the food industry and pharmaceutics has not been reported.

Alginates are natural polysaccharides extracted from brownseaweed. They are anionic compounds, and one of their mostimportant features is the capability to form hydrogels in the pres-ence of divalent cations such as Ca2+. Alginate microparticles act asadjuvants, and vaccine-containing alginate microspheres are effec-

tive for mucosal vaccination in animal species (Rebelatto et al.,2001). However, the use of alginate microparticles for oral vaccinedelivery has been reported to cause a burst effect due to the macro-porous structure of the microparticles, which causes the antigen

2 urnal of Pharmaceutics 448 (2013) 19– 27

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o be preferentially adsorbed to the particle surface (George andbraham, 2006; Wong et al., 2002; Suksamran et al., 2009). Thisurface adsorption can cause stability problems because processesuch as desorption or the degradation of the antigens by enzymesr acidic substances in the body fluids may occur. These obstaclesay be overcome by coating the microparticles with a copolymer

uch as chitosan.Chitosan (CS) is a copolymer of N-acetyl glucosamine (Glc-

Ac) and glucosamine (GlcN). CS has been extensively studiedor the delivery of therapeutic proteins and antigens, particu-arly via mucosal routes because of its excellent mucoadhesivend absorption-enhancing properties (Van der Lubben et al.,001). Various studies have demonstrated the activation of theendritic cells, macrophages and lymphocytes by CS (Villierst al., 2009). However, the main drawback of CS is its waternsolubility at physiological pHs. Versatility in the physico-hemical properties of CS provides an excellent opportunityo engineer antigen-specific adjuvant/delivery systems. ManyS derivatives have been synthesized to enhance its solubil-

ty, mucoadhesiveness and/or immunostimulatory properties.ecently, our research group successfully synthesized modifiedhitosans, methylated N-(4-N,N-dimethylaminobenzyl) chitosanTM-Bz-CS), methylated N-(4-N,N-dimethylaminocinnamyl) chi-osan (TM-CM-CS) and methylated N-(4-pyridinylmethyl) chitosanTM-Py-CS), which demonstrated mucoadhesive (Sajomsang et al.,009) and in vitro absorption-enhancing properties (Kowapraditt al., 2008; 2010). Moreover, the adjuvant effects of these deriva-ives given via the oral route were studied in mice. The resultsemonstrated that the adjuvant activity of TM65CM50CS exhibitedhe greatest immune responses compared with other modified CSsSuksamran et al., 2012).

In previous studies, calcium-alginate microparticles, whichere successfully prepared using an electrospraying technique,

onfirmed their feasibility for delivery of the model protein bovineerum albumin (BSA) in an in vitro release study (Suksamran et al.,009). In this study, calcium-alginate was mixed with yam starcho generate a suitable model antigen carrier. The aim of the presenttudy was to further investigate the feasibility of applying a modi-ed CS, TM65CM50CS, as a surface coating for calcium-alginate andalcium-alginate-yam microparticles to induce immune responseso the model antigen ovalbumin (OVA) administered via the oraloute. The physical and morphological properties, entrapmentfficiency, in vitro release behavior and cytotoxicity of the micro-articles were investigated. In vivo oral immunization in mice waslso investigated.

. Materials and methods

.1. Materials

Low viscosity sodium alginate derived from brown algae (ALV,50 cps) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-olium bromide (MTT) were purchased from Sigma-Chemical Co.St. Louis, MO, USA). The yam starch was extracted from D. esculentas previously described (Nattapulwat et al., 2009). Trimethyl N-(4-,N-dimethylamino cinnamyl) chitosan (TM65CM50CS) productionas carried out according to a previously published procedure

Kowapradit et al., 2010). Trisodium citrate, sodium hydroxide, cal-ium carbonate, ethylenediaminetetraacetic acid (EDTA), napthollue calcium chloride dehydrate and trisodium trimetaphosphateSTMP) were purchased from Ajax Chemicals (New South Wales,ustralia). The Caco-2 cell line was obtained from American

ype Culture Collection (Rockville, MD, USA). Dulbecco’s modifiedagle’s medium (DMEM), trypsin-EDTA, penicillin-streptomycinntibiotics and fetal bovine serum (FBS) were obtained from GIBCO-nvitrogen (Grand Island, NY, USA).

Fig. 1. Production scheme for fabrication of microparticles by an electrosprayingtechnique.

Aluminum hydroxide gel (alum), albumin from chicken eggwhite (ovalbumin [OVA], Grade V, MW = 44 kDa), monoclonal anti-chicken egg albumin (mAb to OVA), IgA kappa, goat anti-mouse IgA(�-chain specific) and rabbit anti-goat IgG peroxidase conjugatewere purchased from Sigma–Aldrich (St. Louis, USA). 3,3′,5,5′-Tetramethylbenzidine (TMB) was supplied by Zymed (Invitrogen,San Francisco, USA). Tween 20 and sulfuric acid (H2SO4, 98%w/w) were obtained from BDH (England). Goat anti-mouse IgGhorse radish peroxidase (HRP) conjugate was ZyMaxTM Grade.Sodium bicarbonate (NaHCO3) was purchased from Fisher Scien-tific (Leicestershire, England). All other chemicals were molecularbiology quality.

2.2. Preparation of OVA-loaded microparticles

The preparation of calcium-alginate microparticles (MP) usingelectrohydrodynamic spraying (EHDA) was carried out accordingto a previously reported protocol (Suksamran et al., 2009). Briefly,alginate and CaCl2 powders were weighed out separately and dis-solved in water under magnetic stirring to prepare stock solutionsof 1% (w/v) alginate and 4% (w/v) CaCl2. To prepare alginate/OVAsolutions, freshly prepared stock solutions of each compound weregently mixed to obtain a homogeneous final solution. Then, 10 mlmixed alginate and OVA solutions (10, 20, 40% w/w to polymer)were extruded dropwise through a 20-gauge blunt needle cappedonto a glass syringe (internal needle diameter 0.9 mm) into 150 mlof 4% (w/v) CaCl2 solution. A voltage of 18 kV was applied to thesetup, and the distance between tip to collector and the feeding ratewere fixed at 30 cm and 1 ml h−1, respectively (Fig. 1). The micro-particles were collected in the CaCl2 solution with gentle stirringfor 30 min. The particles were separated from the CaCl2 solutionby centrifugation at 5862 × g for 10 min (Biofuge Stratos SORVALL;KENDRO, USA), washed twice and resuspended in distilled water.This suspension was lyophilized in a freeze dryer (LABCONCO, Free-zone 2.5, USA) for 3 days. The microparticles were then kept in glassvials at 4 ◦C. The blank microparticles were also prepared in thesame manner without the addition of OVA.

The calcium-alginate-yam microparticles (YMP) were preparedaccording to the same procedure as for calcium-alginate micro-particles described above. Only 0.5% (w/v) of yam starch solutionwas added to the mixture of 1% (w/v) of alginate/OVA (10, 20 and40%, w/w to polymer).

2.3. Preparation of the modified CS-coated microparticles

The microparticles were resuspended in a 0.1% (w/v)TM65CM50CS solution and stirred with a magnetic stirrer at 25 ◦Cfor 2 h. The TM65CM50CS-coated microparticles were separated

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rom the TM65CM50CS solution by centrifugation at 5862 × g for0 min, washed twice and resuspended in distilled water. This sus-ension was lyophilized in a freeze dryer (LABCONCO, Freezone 2.5,SA) for 3 days. The coated microparticles were then kept at 4 ◦C.

.4. Physical characterizations

Morphological characterization of the microparticles waserformed using an inverted microscope (Inverted Research Micro-cope; ECLIPSE TE 2000-U, Japan) at 20x magnification and using acanning electron microscope (SEM; S-3400N, Hitachi, Japan). Thearticle size and Zeta potential analysis were measured using aetasizer Nano ZS (Malvern Instruments, Malvern, UK).

.5. Swelling measurements

The swelling of the microparticles was conducted in 0.1 N HClolution (pH 1.2) and phosphate buffer solution (PBS), pH 7.4.riefly, the diameters of the dried microparticles before and after

ncubation with gastric solution pH 1.2 or PBS pH 7.4 for 0.5, 1, 2, and 5 h were measured at least 100 particles from the imagesormed in an inverted microscope (ECLIPSE TE 2000-U; Model:-DH Nikon®, Japan). Images were captured with a Nikon DXM200 digital camera, followed by the analysis of the digitalizedhotos using Nikon ACT-1 version 2.62 software. The percentagef swelling at different time intervals was determined by the dif-erence between the diameter of microparticles at time t (Dt) andhe initial time (t = 0 [D0]), as calculated according to the followingq. (1) (Miyazaki et al., 2003):

Swelling = (Dt − D0)D0

× 100 (1)

.6. In vitro mucoadhesion study

The in vitro mucoadhesion assay was carried out using theverted intestinal sac technique (Miyazaki et al., 2003). Five cen-imeter segments of porcine jejunum were everted using a glassod. Ligatures were placed at both ends of the segment. One hun-red milligrams of microparticles was scattered uniformly on theverted sac. Then, the sacs were suspended in 50 ml centrifugeubes containing 20 ml of PBS (pH 7.4) and were incubated at 37 ◦Cnd agitated horizontally. The sacs were taken out of the mediumfter immersion for 0.5, 1, 1.5, 2, or 2.5 h and immediately reposi-ioned in a similar tube containing 20 ml of fresh PBS, and the dried,nbound microparticles were weighed. The adhering percentageas calculated by the following Eq. (2):

Adhering = 100 −(

Wt × 100Wo

)(2)

here Wo is the weight of the microparticles at the initial time andt is the weight of the unbound microparticles at the time t after

ncubation.

.7. Entrapment efficiency

The total content of OVA in the OVA-loaded microparticlesas determined. Accurately weighed amounts (0.1 g) of the driedVA-loaded microparticles were placed in 15 ml centrifuge tubesontaining 2 ml of 2% trisodium citrate buffer, followed by con-inuous shaking in a shaker incubator (Orbital Shaking Incubator

odel: SI4) at 200 rpm until the OVA-loaded microparticles wereotally dissolved. The supernatant was measured using a Bradford

ssay to quantify the protein concentration in solution. In the casef TM65CM50CS-coated microparticles, dried TM65CM50CS-coatedicroparticles were dissolved by PBS, pH 7.4, and the supernatantas measured using a Lowry assay. The absorbance at 550 nm

of Pharmaceutics 448 (2013) 19– 27 21

of each sample was measured using a microplate reader (FusionUniversal Microplate Analyser Model: A153601). The entrapmentefficiency percentage and OVA content were calculated using Eqs.(3) and (4), respectively:

%Entrapment efficiency =(

Pt

Lt

)× 100 (3)

where Pt is the amount of OVA embedded in the microparticles,and Lt is the theoretical amount of OVA (obtained from the feedingcondition) incorporated into the microparticles.

OVA content = Pt(mg)Mt(g)

(4)

where Pt is the amount of OVA embedded in microparticles, and Mt

is the total amount of microparticles harvested.

2.8. In vitro release

In vitro release studies were performed by suspending 10 mgof the microparticles in 1.5 ml microcentrifuge tubes with 1 mlof PBS (pH 7.4) or in 0.1 N HCl solution (pH 1.2). All tubeswere then incubated at 37 ◦C under shaking at 200 rpm to main-tain the particles in suspension. To determine the amount ofOVA-loaded microparticles released after a given time, the sam-ple was centrifuged for 15 min at 1077 × g. The absorbance at550 nm of the supernatant was measured in triplicate using amicroplate reader (Fusion Universal Microplate Analyser Model:A153601).

2.9. Evaluation of cytotoxicity

The cytotoxic effects of the microparticles were investi-gated with Caco-2 cells using an MTT cytotoxicity assay. TheCaco-2 cells were maintained in Dulbecco’s modified Eagle’smedium (DMEM), pH 7.4, supplemented with 10% fetal bovineserum, 2 mM l-glutamine, 1% non-essential amino acids and 0.1%penicillin–streptomycin in a humidified atmosphere (5% CO2, 95%air, 37 ◦C). The cells were grown under standard conditions until60–70% confluency. Then, the cells were seeded at a densityof 2 × 104 cells/well in 96-well cell culture plates. After pre-incubation for 24 h, the cells were treated with microparticles atvarious concentrations ranging from 0.01 to 20 mg/ml in serum-free medium (pH 7.4) and incubated for 24 h. After treatment, freshmedium was added, and the cells were incubated for 4 h. Finally,the cells were incubated with 100 �l MTT-containing medium(0.1 mg/ml MTT in serum-free medium) for 4 h. Next, the mediumwas removed, and the formazan crystals that formed in the livingcells were dissolved in 100 �l DMSO per well. The relative via-bility (%) was calculated based on absorbance at 550 nm using amicroplate reader (Universal Microplate Analyzer, Model AOPUS01and AI53601, Packard BioScience, CT, USA). The viability of non-treated control cells was arbitrarily defined as 100%. The relativecell viability was calculated according to Eq. (5), and the IC50was calculated as the microparticle concentration that inhibitedthe growth of 50% of the cells relative to the nontreated controlcells.

Realative cell viability(%) =[OD550,sample − OD550,blank

][OD550,control − OD550,blank

] × 100

(5)

2.10. Oral immunization

Female BALB/c mice, 6–8 weeks of age at the beginning of theexperiment, were obtained from the National Laboratory AnimalCenter, Mahidol University, Thailand. The animals were housed

22 T. Suksamran et al. / International Journal

Table 1Immunization formulations in mice groups 1–5, P and N.

Mice groups Formulations Administration

P 100 �g of OVA in PBS, pH 7.4, with Al(OH)3 S.C.N 250 �g of OVA in PBS, pH 7.4 P.O.1 250 �g of OVA in 0.1% (w/v) TM65CM50CS

solutionP.O.

2 250 �g of OVA in Ca-alginate microparticles(OVA-MP)

P.O.

3 250 �g of OVA in Ca-alginate-yammicroparticles (OVA-YMP)

P.O.

4 250 �g of OVA in TM65CM50CS-coatedCa-alginate microparticles (CS-OVA-MP)

P.O.

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t a controlled temperature with free access to rodent chow andater. This study was approved by an Investigational Review Board

Animal Studies Ethics Committee, Faculty of Pharmacy, Silpakornniversity, Thailand, Approval No. 2-2011). The mice were divided

nto 7 groups, and each group consisted of six mice. The mice weremmunized with various formulations, as shown in Table 1. The

ice in all groups were immunized on days 0 and 14. For group–5 and N, the dose volume was 500 �l and contained 250 �g ofVA. For the positive control group (group P), the mice were immu-ized subcutaneously (s.c.) in the neck region with 200 �l of alumontaining 100 �g of OVA.

.11. Sample collection

On day 0, blood samples (ca. 0.2 ml per animal) were collectedrom the cut tail tip. However, at the end of the study (day 21), thelood (ca. 0.6–1 ml per animal) was collected by cardiac punctureollowing anesthesia of the mice with diethyl ether. The blood sam-les were allowed to clot overnight and were then centrifuged at000 × g for 5 min at room temperature. For tail bleeds, the serumas collected and pooled for each group of mice. For blood col-

ected by cardiac puncture, the serum from each mouse was storedeparately. All serum samples were stored at -20 ◦C until assayed.

To collect fecal samples, the mice were housed separately. Freshecal samples (ca. 6–8 pieces per animal) were collected at the sameime as the blood samples. The fecal samples were stored at -20 ◦Cntil assayed.

.12. Determination of immune responses

The immune response to OVA in various formulations was ana-yzed by an enzyme-linked immunosorbent assay (ELISA). Theevels of OVA-specific serum immunoglobulin G (IgG) antibodynd OVA-specific serum immunoglobulin A (IgA) were determinedPitaksuteepong, 2003) as follows:

.13. Determination of OVA-specific serum immunoglobulin GIgG) antibody

The serum samples were assayed for IgG by ELISA using a6-well flat bottom MaxiSorp NUNC-ImmunoTM plate, and thebsorbance was measured at 450 nm using a microplate readerUniversal Microplate Analyzer, Model AOPUS01 and AI53601,ackard BioScience, CT, USA).

.14. Determination of OVA-specific serum immunoglobulin A

IgA) antibody

Following vacuum drying of the frozen fecal samples using areeze dryer (LABCONCO, Freezone 2.5, USA), the secretory IgA

of Pharmaceutics 448 (2013) 19– 27

(sIgA) was extracted from the fecal samples by adding PBS at aratio of 15 �l/mg dry feces, and the samples were then exten-sively vortexed. Subsequently, the suspensions were centrifugedat 12,000 × g for 20 min. The clear supernatants were assayedfor sIgA by ELISA using a flat bottom 96-well MaxiSorp NUNC-ImmunoTM plate, and the absorbance was measured at 450 nmusing a microplate reader (Universal Microplate Analyzer, ModelAOPUS01 and AI53601, Packard BioScience, CT, USA).

2.15. Statistical analysis

All experimental measurements were collected in triplicate. Thevalues are expressed as the mean ± standard deviation (SD). Thestatistical significance of the differences in each experiment wasexamined using one-way analysis of variance (ANOVA), followedby a least significant difference (LSD) post hoc test. The significancelevel was set at p < 0.05.

3. Results and discussion

3.1. Preparation of microparticles

Recently, electrospinning and electrospraying techniques havebeen developed for the fabrication of fibers and particles for var-ious applications. Previously, we successfully used a high-voltagepower supply to fabricate microparticles through electrospinning.The microparticles consisted of sodium alginate crosslinked withdivalent cations to encapsulate and immobilize bovine serum albu-min (BSA) for the controlled release of the incorporated proteins(Suksamran et al., 2009). This electrospraying technique was iden-tical to the setup used for electrospinning of fibers except that thefiber collector was replaced with a calcium chloride solution jar. Ahigh voltage was used to create an electrically charged jet of poly-mer solution. One electrode was placed into the alginate solution,and the other one was attached to a collector (calcium chloridesolution) (Fig. 1). In this process, the liquid flowing out from the cap-illary nozzle was maintained at a high potential and was subjectedto an electric field, which caused elongation of the meniscus toform a jet or spindle. The jet deformed and disrupted into droplets,mainly due to the electrical force. In this study, Ca-alginate micro-particles (MP) and Ca-alginate-yam microparticles (YMP) wereprepared by crosslinking alginate or alginate-yam, respectively,with calcium chloride solution. OVA was loaded in both micro-particles and the particles were then coated with TM65CM50CS.

3.2. Entrapment efficiency and OVA content

The entrapment efficiency and OVA content of the microparti-cle preparations were investigated. Various initial amounts of OVA(10, 20, 40%, w/w to polymer) were incorporated into the micro-particles. Fig. 2 shows the effects that the various initial amounts ofOVA had on the entrapment efficiency (%EE) and the OVA contentlevel in the Ca-alginate microparticles (MP) and Ca-alginate-yammicroparticles (YMP). For the MP (Fig. 2a), increasing the initialamount of OVA resulted in an overall increase in the amount of pro-tein entrapped in the MP, with the 20% (w/w) initial OVA sampledemonstrating the highest entrapment efficiency and OVA content(40.95 ± 0.4% and 33.21 ± 0.1 mg/g, respectively). After that sam-ple, the amount of protein entrapped in the MP decreased. For theYMP (Fig. 2b), the entrapment efficiency percentage (%EE) and OVAcontent increased from 25% to 36% and from 19.02 to 36.47 mg/g,

respectively, as the initial amount of OVA increased from 10% to 20%w/w. YMP with an initial 20% (w/w) OVA demonstrated the highestentrapment efficiency and amount of OVA content (36.61 ± 0.4%and 36.47 ± 0.1 mg/g, respectively). Therefore, the proper loading

T. Suksamran et al. / International Journal

Initial O VA (% w/w to pol ymer )

Ent

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EE

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Con

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VA

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Initial O VA (% w/w to pol ymer )

10 20 40

10 20 40

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20

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50

60

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ies (% EE) and (�) the OVA content in (a) Ca-alginate microparticles (MP) and (b)a-alginate-yam microparticles (YMP). Each point represents the mean of threexperiments.

f OVA 20% w/w to polymer was chosen to formulate the OVA-oaded microparticles used in the TM65CM50CS-coated OVA-MPnd OVA-YMP.

.3. Physical characterizations

Table 2 summarizes the particle sizes and Zeta potentials ofhe microparticles. The results showed that blank and OVA-loadeda-alginate microparticles (OVA-MP) yielded small particles of.79 ± 0.11 and 0.81 ± 0.12 �m, respectively, with a negative charge-7.16 and -13.6 mV, respectively). In the case of the Ca-alginate-am microparticles (YMP), the particles size appeared to be larger

han those of the MP. However, the size of YMP was still withinn acceptable range size (1–5 �m) for the uptake and transloca-ion of the vaccine via the oral route (Rice-Ficht et al., 2010). Thelank and OVA-loaded Ca-alginate-yam microparticles (OVA-YMP)

able 2article size and Zeta potential of microparticles with and without OVA.

Samples code Mean size(�m ± SD)

Zeta potential(mV ± SD)

Ca-alginate microparticles Blank-MP 0.79 ± 0.11 −7.16 ± 0.6OVA-loaded Ca-alginate

microparticlesOVA-MP 0.81 ± 0.12 −13.60 ± 0.5

TM65CM50CS-coatedOVA-loaded Ca-alginatemicroparticles

CS-OVA-MP 1.49 ± 0.11 10.93 ± 0.4

Ca-alginate-yammicroparticles

Blank-YMP 1.79 ± 0.13 −12.00 ± 0.5

OVA-loaded Ca-alginate-yammicroparticles

OVA-YMP 1.69 ± 0.14 −15.90 ± 0.8

TM65CM50CS-coatedOVA-loadedCa-alginate-yammicroparticles

CS-OVA-YMP 2.96 ± 0.23 6.03 ± 0.7

of Pharmaceutics 448 (2013) 19– 27 23

yielded particles of 1.79 ± 0.13 and 1.69 ± 0.14 �m, respectively,with a more negative charge (-12 and -15.9 mV, respectively). Theaddition of 0.5% (w/v) of yam starch to alginate solution resultingin an increase of the particle size of YMP could be attributed tothe increase of viscosity of dispersed phase which led to difficul-ties for breaking up dispersed phase into smaller droplets. Similarresults have been reported by other researchers (Li et al., 2009).After the incorporation of OVA, a more negative Zeta potential wasobserved with both MP and YMP. This result was likely due to thenegative charge of OVA. This result corresponded with a previousreport by San Roman et al. (2008), who found that the Zeta poten-tial shifted negatively when CpG motifs were encapsulated. In thecase of coated microparticles, TM65CM50CS was selected because ofits high mucoadhesive property (Sajomsang et al., 2009) and adju-vant activity (Suksamran et al., 2012). The coating efficiencies of themicroparticles were determined from the net charges produced bythe microparticle coats, as determined using a Zeta potential ana-lyzer. For instance, while the prepared microparticles possessed anaverage negative charge, the microparticles completely coated withthe modified chitosan were expected to exhibit a positive charge.TM65CM50CS-coated OVA-loaded Ca-alginate microparticles (CS-OVA-MP) and TM65CM50CS-coated OVA-loaded Ca-alginate-yammicroparticles (CS-OVA-YMP) were of spherical shape with meandiameters of 1.49 ± 0.11 and 2.96 ± 0.23 �m, respectively, and pos-itive surface charges (+10.93 and +6.03 mV, respectively). Fig. 3shows the morphology obtained from an inverted-microscope at200X magnification (Fig. 3a, c, d and f) and SEM images at 500X mag-nification (Fig. 3b and e) of CS-OVA-MP (upper) and CS-OVA-YMP(lower). The inverted-microscope images suggest that the parti-cles are in gel state, almost transparent, spherical, smooth andhomogeneously distributed, without evidence of collapsed parti-cles. However, after freeze drying, the particles are in solid stateand are appeared to possess a withered shape (SEM images). Theseresults could be explained by the dehydration of the microparticlesduring lyophilization (Kasper and Friess, 2011). After rehydrationof the microparticles with distilled water, similar mean diameterswere obtained as before freeze drying (Fig. 3c and f) and were ofspherical shape.

3.4. Swelling properties

The percentage of swelling at different times was determinedby calculating the diameter of microparticles before and after incu-bation in 0.1 N HCl solution (pH 1.2) or phosphate buffer solution(PBS), pH 7.4. The results revealed that all of the formulations incu-bated with gastric pH 1.2 exhibited a lower percent swelling thanincubated with PBS pH 7.4 (Fig. 4). All formulations swelled rapidlywhen immersed in phosphate buffer, especially the TM65CM50CS-coated Ca-alginate microparticle (CS-MP) formulation (Fig. 4b). TheCS-MP formulation exhibited the highest percent swelling at 0.5,1 and 2 h, as compared with the other formulations. After a 5-hincubation, the percent swellings of MP, CS-MP, YMP and CS-YMPwere 83.21 ± 28%, 110.97 ± 24%, 100.70 ± 30% and 109.89 ± 15%,respectively. Increasing the time of incubation increased the per-centage of swelling. The adhesive properties and cohesiveness ofmucoadhesive polymers are reported to be generally affected bytheir swelling behavior (Mortazavi and Smart, 1993). Mucoad-hesive microparticles are predicted to take up water from theunderlying mucosal tissue by absorbing, swelling, and capillaryeffects, leading to considerably stronger adhesion (Duchene andPonchel, 1992). The considerable increase in the swelling of micro-particles might have been due to the swelling force resulting from

the presence of counterions that neutralized the carboxylic groupsof alginate present in neutral media (Tavakol, 2009). The anionicnature of alginate might have enhanced the repulsion among themolecular chains and resulted in an increase in the swelling ratio.

24 T. Suksamran et al. / International Journal of Pharmaceutics 448 (2013) 19– 27

Fig. 3. The morphology of (upper) CS-OVA-MP and (lower) CS-OVA-YMP as visualized by an inverted microscope at 200x magnification before (a, d) and after freeze dryingby a scanning electron microscope (b, e) at a magnification of 500x, and rehydration with distilled water by an inverted microscope at 200x magnification (c, f).

Fig. 4. The percent swelling of Ca-alginate microparticles (MP), TM65CM50CS-coated Ca-alginate microparticles (CS-MP), Ca-alginate-yam microparticles (YMP) andTM65CM50CS-coated Ca-alginate-yam microparticles (CS-YMP) at different incubation times conducted in (a) 0.1 N HCl solution (pH 1.2) and (b) phosphate buffer solution(PBS), pH 7.4.The data are presented as the mean ± SD (n = 100). (*) indicates p < 0.05.

T. Suksamran et al. / International Journal of Pharmaceutics 448 (2013) 19– 27 25

0

10

20

30

40

50

60

70

80

90

100

0 0.5 1 1.5 2 2.5

Perc

enta

ge o

f ad

heri

ng

Time (h)

* *

* ***

*

Fig. 5. In vitro mucoadhesion of microparticles to porcine small intestines: (�)Ca-alginate microparticles (MP), (�) Ca-alginate-yam microparticles (YMP), (�)Tco

AcsbIapwb(

3

bmpstdsrarCcoMrftpepsssnpbrthtt

*

*

Time (h)

Cum

ulat

ive

rele

ase

(%)

20

60

0

40

80

100

100 5 20 2515

Fig. 6. Release profile of OVA from (�) OVA-MP, (�) OVA-YMP (�) CS-OVA-MP and

The release profiles of OVA from the OVA-loaded microparticlesare shown in Fig. 6. The release profile of OVA-MP indicatedhigher release than CS-OVA-MP. These results suggest that theOVA entrapped in the CS-MP was mostly retained under these

0

50

100

0.01 0.1 1 10 100

Rel

ativ

e ce

ll vi

abili

ty (

%)

Concentration (mg/ml)

M65CM50CS-coated Ca-alginate microparticles (CS-MP), and (�) TM65CM50CS-oated Ca-alginate-yam microparticles (CS-YMP). Each point represents the meanf three experiments. (*) indicates p < 0.05.

fter adding yam starch into the Ca-alginate formulation, the per-ent swelling was increased due to the hydrophilic nature of thetarch, which imparts increasing hydrophilicity to the blend andrings about an increase in the swelling ratio (Roy et al., 2009).n the case of the TM65CM50CS-coated microparticles, the degreend rate of swelling were higher than those of uncoated micro-articles. This effect might have resulted from the swelling of theater-soluble TM65CM50CS at pH 7.4 due to hydrogen bonding

etween the amino and hydroxyl groups within the chitosan chainsRohindra et al., 2004).

.5. In vitro mucoadhesion study

The mucoadhesive properties of microparticles were evaluatedy everted sac experiments using porcine small intestine. Thisethod determined the unbound microparticles and the results are

resented as adherence percentages. A high percentage of adhe-ion indicates that microspheres exhibit excellent mucoadhesiono mucosal tissue and also directly indicated the prolonged resi-ence time in the intestine. Fig. 5 shows the results of the evertedac experiments for the different formulation microparticles. Theesults demonstrated that the uncoated microparticles rapidly sep-rated; the adherence of MP and YMP at 1 h was 29.62% and 11.29%,espectively. In contrast, the coated microparticles CS-MP andS-YMP separated from the sacs gradually, exhibiting adhering per-entages at 1 h of 45.64% and 43.38%, respectively. The adherencesf the microparticles after 2 h of incubation were as follows: CS-P (43.60%) > CS-YMP (39.66%) > MP (31.53%) > YMP (5.71%). These

esults indicated that the mucoadhesive ability was affected by sur-ace coating with TM65CM50CS. Generally, chitosan has been showno interact with mucin (Fiebrig et al., 1995). Takeuchi et al. (1996)repared liposomes coated with chitosan and found that theyxhibited a prolonged residence time in the GI tract of rats in com-arison to uncoated liposomes. The adhesive properties and cohe-iveness of chitosan are reported to be generally affected by theirwelling behavior (Mortazavi and Smart, 1993). In the mucoadhe-ion process, the swelling and expansion of the polymer chain isecessary because the interpenetration and entanglement of theolymers and the mucous networks are considered to be responsi-le for adhesion. Therefore, bioadhesives should swell and expandapidly when they come in contact with water. In addition, the chi-

osan derivative TM65CM50CS is a positive polymer, with numerousydrophilic functional groups such as hydroxyl groups in the chi-osan molecules that have an ability to form hydrogen bonds withhe mucus molecules. This interaction is reported to be responsible

(�) CS-OVA-YMP incubated with 0.1 N HCl (pH 1.2) for 2 h and then replaced withPBS (pH 7.4) until 24 h. Each point represents the mean of three experiments. (*)indicates p < 0.05.

for the mucoadhesive property of this polymer. In contrast, the MPand YMP microparticles possessed a negative charge. In the pres-ence of the PBS buffer (pH 7.4), this negative charge could havebeen repelled by the negatively charged mucus, leading to poormucoadhesion. Dhaliwal et al. (2008) investigated the mucoadhe-sivity of different microsphere formulations in pig intestine andreported that strong electrostatic attraction between mucin andcarbopol 71G or chitosan appears to contribute to good mucoadhe-sion. These previous and current findings suggest that TM65CM50CScan improve the bioadhesive properties of the fabricated micro-particles. The good mucoadhesive properties lead to increasedresidence time of the device in the gastrointestinal tract, which willmost likely relate to increased bioavailability of the encapsulateddrug (Helliwell, 1993; Miyazaki et al., 2003) or enhanced mucosalimmune response of encapsulated antigen (Islam et al., 2011).

3.6. In vitro release from OVA-loaded microparticles

Fig. 7. Cell viabilities of Caco-2 cells incubated with (�) Ca-alginate micro-particles (MP), (�) Ca-alginate-yam microparticles (YMP), (�) TM65CM50CS-coatedCa-alginate microparticles (CS-MP), or (�) TM65CM50CS-coated Ca-alginate-yammicroparticles (CS-YMP) at concentrations 0.01–20 mg/ml in pH 7.4 medium for24 h. Each value represents the mean ± SD of eight wells.

26 T. Suksamran et al. / International Journal of Pharmaceutics 448 (2013) 19– 27

Fig. 8. Antibody titers of (a) serum IgG and (b) secretory IgA obtained from mice following oral immunization with OVA in PBS (pH 7.4) (group N); OVA in 0.1% (w/v) ofT ), OVA( rs indi

cYtawOppapwimMshws

3

uviTwatlpti2(mcm

3

pqca

M65CM50CS solution (group 1); or OVA-loaded microparticles: OVA-MP (group 2group P) administered subcutaneously was used as a positive control. The white bandicates p < 0.05.

onditions. This result correlated with the release profile of OVA-MP, which exhibited higher release than did CS-OVA-YMP. Theotal release of OVA from the uncoated microparticles (OVA-MPnd OVA-YMP) was approximately 60% and 57%, respectively,hereas that from the coated microparticles (CS-OVA-MP and CS-VA-YMP) was approximately 35% and 25%, respectively, over aeriod of 24 h. All of the formulations exhibited slow release at lowH, likely due to the limited swelling of alginate microparticlest low pH that resulted in decreased OVA release from micro-articles. The release of OVA from the uncoated microparticlesas higher than that from the coated microparticles and exhib-

ted sustained release for 24 h. This result may be due to theacroporous structure and rapid dissolution at intestinal pH ofP and YMP, which may cause increased release of the core

ubstances than is observed with coated particles (Georg and Abra-am, 2006). Moreover, at pH 7.4, the uncoated microparticlesere likely eroded due to the properties of alginate and native

tarch.

.7. Cytotoxicity of microparticles

The cytotoxicity effects of the TM65CM50CS-coated andncoated microparticles in Caco-2 cells were determined as % celliability, as shown in Fig. 7. A concentration-dependent cytotoxic-ty of the microparticles was observed for a 24-h incubation period.he IC50 values, indicating 50% cell deactivation, of all formulationsere greater than 20 mg/ml, indicating that no toxicity occurred

fter incubation with microparticles for 24 h. These results suggesthat all microparticle formulations prepared in this study were safe,ikely due to the nature of alginate and starch, which are naturalolymers. The findings correlated with the results of other studieshat indicated that both alginate and starch microparticles exhib-ted noncytotoxic behavior (Silva et al., 2004; Suksamran et al.,011). The TM65CM50CS solution demonstrated a slight toxicityKowapradit et al., 2010), but when TM65CM50CS was coated onto

icroparticles, there was no cytotoxicity observed at the sameoncentrations. The results confirm that the coated and uncoatedicroparticles were safe in vitro.

.8. In vivo immunological study

Serum IgG antibody responses to entrapped OVA in micro-

articles produced from MP, CS-MP, YMP and CS-YMP wereuantified (Fig. 8a). The results indicated that all mice vac-inated with OVA-loaded microparticles produced a strongnd significant enhancement in IgG titers against OVA. Only

-YMP (group 3), CS-OVA-MP (group 4), and CS-OVA-YMP (group 5). OVA in alumicate antibody titers at day 0, while black bars indicate antibody titers at day 21. (*)

minimal immune responses were present in the groups vacci-nated with OVA in PBS. The IgG levels of mice that receivedOVA-loaded, TM65CM50CS-coated microparticles (CS-OVA-MP andCS-OVA-YMP) were significantly higher than those that receivedOVA-loaded, uncoated microparticles (OVA-MP and OVA-YMP).Similar to the IgG responses, the IgA levels observed in the micetreated with OVA in coated microparticles were significantly higherthose vaccinated with OVA in uncoated microparticles (Fig. 8b).The level of the IgA response obtained from mice immunized withCS-OVA-MP was slightly higher than the CS-OVA-YMP group. Inaddition, the mice that received OVA entrapped within micro-particles exhibited higher IgA responses than mice receiving OVAsolution (group N and 1); there was a very low IgA responseobserved with mice immunized s.c. with OVA in alum (group P).This result suggested that oral vaccine delivery can induce botha systemic and mucosal immune response. The association of thevaccine with microparticulate drug carrier systems may preventits degradation in the stomach and the gut and may stimulatethe M-cells to transport the vaccine to the dome of the Peyer’spatches. After transportation to the dome of the Peyer’s patches,the microparticles are degraded and the vaccine is released intothe lymphoid tissue, resulting in an immune response (Alpar et al.,2000; Barackman et al., 1998).

4. Conclusion

Microparticles from calcium-alginate (MP), TM65CM50CS-coated calcium-alginate (CS-MP), calcium-yam-alginate (YMP)and TM65CM50CS-coated calcium-yam-alginate (CS-YMP) weresuccessfully prepared by an ionotropic gelation method usingan electrospraying technique. The size of the microparticleswas 1–3 �m, and the shape was spherical. The coated micro-particles resulted in greater sustained release and mucosal immuneresponse than did uncoated microparticles. From this study, it couldbe concluded that TM65CM50CS-coated microparticles represent auseful carrier to improve the immunogenicity of oral vaccines.

Acknowledgments

The authors wish to thank the Silpakorn University Researchand Development Institute for financial support.

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