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Process Biochemistry 46 (2011) 631–635

Contents lists available at ScienceDirect

Process Biochemistry

journa l homepage: www.e lsev ier .com/ locate /procbio

ffect of microencapsulated precipitants of Lactobacillus casei ATCC 393 onelicobacter pylori eradication

.A. Koa,1, H.J. Lima,b,1, H.J. Parka,c,∗

School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of KoreaCosmetics R&D Center, Woongjin Coway, Seoul 153-792, Republic of KoreaDepartment of Packaging Science, Clemson University, Clemson, SC 29634-0370, USA

r t i c l e i n f o

rticle history:eceived 19 May 2010eceived in revised form 13 October 2010

a b s t r a c t

Precipitants of Lactobacillus casei ATCC 393-loaded chitosan microspheres were prepared by electro-static interaction between chitosan (CS) and poly-�-glutamic acid (�-PGA). The loading efficiency wasapproximately 80%, and the zeta potential of the microspheres became more positively charged as the

ccepted 21 October 2010

eywords:actobacillus casei ATCC 393elicobacter pylorihitosan

molar ratio of the chitosan amino group was increased. The particles were confirmed to have a sphericalshape by optical microscopy. A release test using simulated gastric fluid (SGF) was also performed. Theresults show that the precipitants were gradually released from the CS-�-PGA microspheres at a rate ofalmost 80% after 60 min. Both the inhibition of Helicobacter pylori growth and the anti-adhesive activity ofencapsulated precipitants were maintained after exposure to SGF. These results indicate that CS-�-PGA

e a d

oly-�-glutamicicrospheres

microspheres may provid

. Introduction

Helicobacter pylori is the main etiologic factor in the develop-ent of gastritis, gastric ulcers, and gastric carcinoma. H. pylori

s considered a Class I (definite) biological carcinogen in humansy the International Agency for Cancer Research and the Worldealth Organization (WHO) [1]. H. pylori resides in the stomach,here it attaches to the gastric epithelium or the interface between

he mucus layer and the apical surface of epithelial cells [2]. Theevelopment of resistance to commonly used antibiotics, such aslarithromycin, and metronidazole, by H. pylori is a major cause ofreatment failure [3]. The inadequacy of antibiotics as an ongoinglinical treatment increased the need for effective alternatives suchs natural anti-microbial proteins and peptides.

Probiotics are live microbial food ingredients that have benefi-ial effects on human health by increasing humoral immunity andmproving the balance of intestinal microbiota. Lactic acid bacteria

LAB) are commonly incorporated into foods for use as probi-tics [4]. These organisms alter the composition of gastrointestinalora by producing lactic acid, bacteriocins, and antimicrobial pep-ides, which together inhibit pathogen activities. Bacteriocins, or

∗ Corresponding author at: School of Life Sciences and Biotechnology, Korea Uni-ersity, 1,5-Ga, Anam-Dong, Sungbuk-Gu, Seoul 136-701, Republic of Korea.el.: +82 2 3290 3450; fax: +82 2 953 5892.

E-mail address: hjpark@korea.ac.kr (H.J. Park).1 Both equally contributed to this research as co-first authors.

359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2010.10.012

elivery system for the effective eradication of H. pylori.© 2010 Elsevier Ltd. All rights reserved.

bacteriocin-like substances, are peptides or proteins that exhibitinhibitory activity against sensitive strains of bacteria [5]. However,these unstable compounds need to be protected from degradation,such as under low pH conditions. Therefore, microencapsulationtechniques involving a core surrounded by a thin membrane barrierhave been investigated for the improvement of probiotic viabil-ity.

In order to develop an oral delivery system, both residence timein the gastrointestinal (GI) tract and release rate of the active ingre-dient must be optimized [6]. One of the most extensively studiedmethods for prolonging residence time in the GI tract is based onthe use of mucoadhesive polymers that adhere to the mucus layerand then release the loaded drug in a sustained manner [7]. Anyintimate contact with the mucosal surface can result in increasedretention time and an increased concentration of core materials inthe GI tract. Among the encapsulation devices, chitosan micropar-ticles have been widely studied as mucoadhesive materials thatenhance the penetration of macromolecules across the intestinaland nasal barriers [8,9].

Chitosan [poly(�-(1 → 4)-2-amino-2-deoxy-d-glucose)], a nat-ural cationic polysaccharide derived from chitin, has been studiedby the pharmaceutical industry as carrier for drugs and proteins[10]. Chitosan may enhance absorption by allowing prolonged

interaction between the delivered drug and the membrane epithe-lia. The charged amino group of d-glucosamine within chitosancan interact electrostatically with sialic acid (N-acetyl nuraminicacid) in the gastric mucus [8]. Therefore, chitosan microspherescan improve the gastric residence time of core materials and also

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32 J.A. Ko et al. / Process Bio

rovide a pH-responsive release profile by swelling in the acidicastric environment [9].

Chitosan microspheres were prepared by simple ionic-gelationethods with poly-�-glutamic acid (�-PGA). �-PGA is hydrophilic

nd polyanion. It can form gel by ionic interaction between pos-tively charged amino groups of chitosan and negatively chargedarboxyl groups of �-PGA. �-PGA is nontoxic, hydrophilic, water-oluble and biodegradable, and has been successfully utilized in theosmetic, food, and medical industries [11,12].

The objective of this study was to investigate the effect ofhitosan (CS)-�-PGA microspheres loaded with precipitants of Lac-obacillus casei ATCC 393 on the eradication of H. pylori. For thisurpose, we prepared microspheres with chitosan as the polyca-ion and �-PGA as the polyanion and tried to control the loadingfficiencies by changing the molar ratio of –NH3

+:–COO−. Also wenvestigated the inhibition of H. pylori growth and the anti-adhesivectivity of encapsulated precipitants.

. Materials and methods

.1. Materials

.1.1. Raw materialsChitosan oligosaccharide with an average viscosity molecular weight of 5 kDa

nd a 90% degree of deacetylation was purchased from Sigma (St. Louis, MO, USA).-PGA (Na salt form, MW. 50 kDa) was purchased from Bioleaders Corp. (Korea).mmonium sulfate (99.0%) was purchased from Samchun Pure Chemical Co. Ltd.

Korea). To prepare phosphate buffered saline (PBS), sodium phosphate (Dibasic,onobasic) was purchased from Duksan Pure Chemical Co. Ltd. (Korea).

.1.2. LAB strain and culture conditionsL. casei ATCC 393 used in this study was sub-cultured initially in 20 ml of MRS

roth (Man Rogosa Sharpe broth) at 37 ◦C for 18 h under anaerobic conditions. Theesulting cultures were transferred into MRS broth and incubated under the sameonditions. Cultures were harvested by centrifugation at 4500 × g at 4 ◦C for 30 min,ollowed by washing with PBS (pH 7.2) and centrifugation as above. The washedacterial cells were mixed with MRS broth, frozen at −70 ◦C for 1 day, and freeze-ried for 72 h in a chamber-type freeze drier (FD5518, Ilshin Lab Co. Ltd., Korea)nder 5 �mHg.

.1.3. H. pylori strain and culture conditionsH. pylori strains (KCTC 12083) were obtained from the Biological Resource Cen-

er, Korea Research Institute of Bioscience and Biotechnology (KRIBB). Brain heartnfusion broth (37 g/L, BHIB, Difco Laboratories, Detroit, USA) containing 10% equineerum and 0.25% yeast extract (Hyclone, Road Logan, UT, USA) was used as liquidedia. The solid medium used was Columbia blood agar, which consisted of 42 g/L

f Columbia Agar (Oxoid Ltd., Basingstoke, England) and 70 ml/L of horse blood.

.2. Methods

.2.1. Precipitation of L. casei ATCC 393 by ammonium sulfatePrecipitants (393 precipitants) of L. casei ATCC 393 spent culture supernatants

SCS) were partially purified by ammonium sulfate precipitation. LAB culture brothas centrifuged at 6000 × g for 30 min at 4 ◦C (Beckman Coulter, Inc., USA). The

upernatant was adjusted to pH 6.5 in order to avoid acid side effects. The SCS wasransferred to a beaker in a cold chamber maintained at 4 ◦C. Ammonium sulfateas then added to a concentration of 60% (w/v) with gentle stirring overnight. Sub-

equently, the mixture was centrifuged at 6000 × g for 30 min at 4 ◦C, and the pelletas dissolved into phosphate buffered saline (PBS, pH 7.2). The 393 precipitantsere freeze-dried (FD5518, Ilshin Lab Co. Ltd., Korea) and stored at −20 ◦C.

.2.2. Well diffusion assayThe growth inhibition effects of LAB-SCS and LAB-SCS-derived 393 precipitants

n H. pylori KCTC 12083 were investigated by the Sgouras method [13]. H. pyloriulture broth was plated (108 CFU/plate) on Columbia blood agar plates containing% horse blood in the absence of antibiotics, and Pasteur pipettes were used toonstruct wells in the agar containing 50 �l of LAB-SCS or precipitants. Plates werencubated under microaerophilic conditions (5% O2, 10% CO2 and 85% N2) at 37 ◦Cor 72 h, and the diameters of the inhibition zones were measured.

.2.3. Preparation of chitosan-�-PGA microspheres

Chitosan-�-PGA (CS-�-PGA) microspheres were produced by the addition of

hitosan solution (with 10% 393 precipitants in 10 ml of PBS (pH 7.2)) into �-PGAqueous solution at varying molar ratios of –NH3

+:–COO− (1:2, 1:1, 1.5:1, 2:1, 2.5:1,nd 3:1). The mixture solution was magnetically stirred for 30 min at room tem-erature. The CS-�-PGA microspheres were collected and washed twice with PBSpH 7.2) by centrifugation at 15,000 rpm for 20 min. Microspheres were dispersed

stry 46 (2011) 631–635

by sonication for 5 min in a sonication bath, followed by freeze-drying for 48 h usinga chamber-type freeze-drier (FD5518, IlshinLab Co. Ltd., Korea).

2.2.4. Characterization of CS-�-PGA microspheresThe surface charge of prepared microspheres was measured using a Nano Zeta-

sizer (Malvern Ltd., Malvern, UK). The folded capillary cell has the dipole connectedto the instrument. The surface charge was obtained by passing an electric currentthrough the samples. The zeta potential values were determined by gauging themoving direction and velocity of the resulting dispersed microspheres in the givenelectric field.

The morphology of 393 precipitants encapsulated in CS-�-PGA microsphereswas examined by optical microscopy (magnification ×200).

2.2.5. Loading efficiencyThe amount of 393 precipitants encapsulated in the CS-�-PGA microspheres

was determined. The CS-�-PGA microspheres containing 393 precipitants were cen-trifuged twice at 15,000 rpm for 30 min after which the supernatants were collected.The concentration of the precipitants in 1 ml of supernatant was assayed by mea-suring the absorbance at 215 nm using a UV spectrophotometer (Shimadzu 1601PC,Japan). Experiments were performed in triplicate (n = 3) and loading efficiencieswere calculated as follows.

Loading efficiency (%) = Calculated 393 precipitants concentrationTheoretical 393 precipitants concentration

× 100

2.2.6. Release test in simulated gastric fluidThe release profile of 393 precipitants was studied under simulated gastric fluid

(SGF) of pH 1.5 without pepsin. CS-�-PGA microspheres (1 g) were added to conicaltubes containing 20 ml of SGF and incubated at 37 ◦C. 1 ml aliquots were removedand samples were assayed for concentration of 393 precipitants at time intervals of0, 10, 20, 30, 40, 50 and 60 min by measuring the absorbance at 215 nm using an UVspectrophotometer (Shimadzu 1601PC, Japan).

2.2.7. Anti-H. pylori effects of microspheres by well diffusion assayOne gram of non-encapsulated 393 precipitants or CS-�-PGA microspheres was

completely dispersed in 10 ml of SGF at 37 ◦C. Samples were periodically removedevery 20 min and centrifuged at 15,000 rpm for 15 min. The pellets were resus-pended in PBS (pH 7.2) and 50 �l aliquots were made.

H. pylori culture broth was plated (108 CFU/plate) on Columbia blood agar platescontaining 7% horse blood in the absence of antibiotics, and wells were made intoagar using Pasteur pipettes in which 50 �l of non-encapsulated or encapsulated393 precipitants treated with SGF. Plates were incubated under microaerophilicconditions (5% O2, 10% CO2, 85% N2) at 37 ◦C for 72 h, and inhibition zone diameterwere measured.

2.2.8. Anti-adhesive effects microspheres on the adhesion of H. pylori to gastriccarcinoma cell line (AGS)

The gastric cancer cell line AGS (ATCC CRL 1739) was incubated in a 12-wellplate (BD science) with RPMI 1640 medium (Gibco BRL) containing 10% fetal bovineserum (FBS) and 1% penicillin–streptomycin (Hyclon). AGS cells cultured for 18 hwere washed three times with RPMI 1640 medium (without serum).

One gram of free 393 precipitants or CS-�-PGA microspheres was completelydispersed in 10 ml of SGF and then sampled at time intervals of 0, 20, 40, and 60 min.After centrifugation at 15,000 rpm for 15 min, pellets were resuspended in PBS (pH7.2) and 500 �l aliquots were sampled. H. pylori (ca., 108 CFU ml−1) was suspendedin the aliquots, which were incubated at 37◦C for 1 h. Treated H. pylori cells werewashed with PBS (pH 7.2) and then resuspended in RPMI 1640 medium (withoutserum). The cell suspension was added to each well of the tissue culture plate, whichwas plated under 10% CO2–90% air at 37 ◦C for 2 h. Subsequently, cells were washedwith PBS to remove unattached H. pylori. AGS cells were treated by 0.1% Triton X-100. H. pylori cells attached to the wells were diluted appropriately followed byplating on antibiotic-selective agar and incubation at 37 ◦C for 4 days. Attached H.pylori cells were quantified by the dilution plate method.

3. Results and discussion

3.1. Anti-H. pylori effects of precipitants of L. casei ATCC 393

L. casei ATCC 393 SCS and precipitants of L. casei ATCC 393(393 precipitants) were examined for growth inhibition of H. pylori(Fig. 1). The average inhibition zones of the 393 precipitants andL. casei ATCC 393 SCS were 7 and 4.3 mm, respectively. It means

that 393 precipitants is more effective than L. casei ATCC 393 SCS inthe growth inhibition of H. pylori. Lactobacilli have been reportedto inhibit the growth of H. pylori as well as attachment to gastricepithelial cell lines [13,14]. In this study, 393 precipitants were usedsince they had higher inhibitory activity on H. pylori.

J.A. Ko et al. / Process Biochemistry 46 (2011) 631–635 633

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casei ATCC 393culture supernatants

ig. 1. In vitro inhibitory effects of cell spent culture supernatants (SCS), and pre-ipitants of L. casei ATCC 393 on the growth of H. pylori KCTC 12083.

.2. Preparation and characterization of CS-�-PGA microspheres

Chitosan microspheres were prepared by ion–ion interactionetween the carboxylic groups of �-PGA and the amino groupsf chitosan. Variations in the zeta potentials of CS-�-PGA micro-pheres were investigated depending on the ratio of chitosan–NH3

+) to �-PGA (–COO−) (data not shown). The zeta poten-ials of samples with a 1:2 ratio of NH3

+:–COO− were negativelyharged (−3.7 mV) due to the presence of carboxyl (–COO−) groupsn the hydrophilic �-PGA shell. Others samples were positivelyharged (average +23.25 mV) due to an increase in the molecu-ar ratio of chitosan. Therefore, the molecular ratio of chitosanffected the surface charge of the microspheres, and the posi-ively charged chitosan microspheres facilitated the mucoadhesiverocess to the negatively charged mucosal surface. The positivelyharged d-glucosamine residues of the chitosan microspheresnteract electrostatically with the negatively charged sialic acid

esidues of mucin in the stomach, resulting in prolonged residenceime [10,15].

Fig. 2 shows the morphology of CS-�-PGA microsphere-ncapsulated 393 precipitants by optical microscopy (×200).icrospheres were uniformly spherical in shape, similar to reports

ig. 2. Optical microscopy (×200) image of CS-�-PGA microsphere-encapsulated93 precipitants. The scale bar means 100 �m.

Fig. 3. Loading efficiency of CS-�-PGA microsphere-encapsulated 393 precipitantswith different molar ratios of NH3

+ on chitosan and –COO− on � -PGA.

that chitosan-�-PGA microspheres are spherical in shape with asmooth surface [11,16].

Fig. 3 shows that the loading efficiencies of CS-�-PGA micro-spheres prepared with various molar ratios of –NH3

+:–COO−

ranged from 76.2 to 82.4%. In addition, the molar ratio of–NH3

+:–COO− did not affect the loading efficiency. Therefore, CS-�-PGA microspheres prepared with a 2:1 molar ratio of –NH3

+:–COO−

were selected arbitrarily and used for the rest of this study.

3.3. In vitro release study

The release profile of 393 precipitants from chitosan micro-spheres in enzyme-free simulated gastric fluid (SGF, pH 1.5) isshown in Fig. 4. Approximately 80% of the loaded 393 precipi-tants were released from the microspheres within 60 min. Thisrelease, specifically, the interaction between self-assembled chi-tosan and the �-PGA polyionic complex, was strongly affected bypH. Under low pH, most carboxyl groups on �-PGA became pro-tonated, which disrupts the complex and leads to release of the

drug from the microspheres [17]. Therefore, microspheres at pH1.5 were dissolved and the majority of entrapped 393 precipitantswere released.

Fig. 4. Percentage of 393 precipitants released from CS-�-PGA microspheres insimulated gastric fluid (SGF, pH 1.5).

634 J.A. Ko et al. / Process Biochemistry 46 (2011) 631–635

Time (min)

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ig. 5. In vitro inhibitory activities of non-encapsulated ( ) and encapsulated

93 precipitants ( ) on the growth of H. pylori KCTC 12083 in simulated gastricuid (SGF, pH 1.5).

Chang et al. [18] prepared chitosan-�-PGA nanoparticles at dif-erent pH values. They reported that chitosan-�-PGA nanoparticlesecame unstable and broke apart at pH 1.2 due to insufficient elec-rostatic interaction between chitosan and �-PGA. Most carboxylicroups of �-PGA were in the form of –COOH.

.4. Growth inhibition effects of 393 precipitants on H. pylori

To determine how pH influences the growth inhibitory activ-ties of 393 precipitants, we investigated the inhibition effects of93 precipitants on H. pylori after treatment with SGF (pH 1.5). Ashown in Fig. 5, the growth inhibition effects of non-encapulated93 precipitants on H. pylori KCTC 12083 were markedly decreasedfter exposure to SGF. Specifically, the effects were decreasedy more than 50% in just 20 min (SGF exposure time), and the

nhibitory zone was completely gone after 60 min. However, therowth inhibition effects of the encapsulated precipitants wereaintained until 60 min due to protection conferred by the CS-�-

GA microspheres against the gastric environment. Fig. 4 showshat the 393 precipitants were gradually released from micro-pheres over 60 min, which coincides with Fig. 5 showing that therowth inhibition effects of encapsulated precipitants were main-ained. Therefore, the CS-�-PGA microspheres were better thanhe non-encapsulated 393 precipitants in sustaining the inhibitoryctivity of 393 precipitants on H. pylori in a gastric environment.

.5. Anti-adhesive effect of 393 precipitants on H. pylori

The attachment of H. pylori to the gastric epithelium is impor-ant for active inflammation of the mucosal layer. H. pylori shows aide spectrum of different specificities regarding adhesion to host

ells [19]. Several surface carbohydrates mediating cell adhesionave been identified. Especially, the Lewis b blood group antigen,hich is typically expressed on human gastric epithelial cell sur-

aces such as AGS cells, is known to mediate the adherence of H.ylori to human gastric mucosa [20].

To investigate the anti-adhesive activity of 393 precipitants, wexamined the inhibitory effects of non-encapsulated and encap-

ulated 393 precipitants in CS-�-PGA microspheres after SGFreatment on the adhesion of H. pylori to AGS cells (Fig. 6).on-encapsulated 393 precipitants (SGF exposure time: 0 min)arkedly reduced the adhesion of H. pylori to AGS cells to 32%. How-

ver, after non-encapsulated 393 precipitants were treated with

Fig. 6. Inhibitory activities of non-encapsulated ( ) and encapsulated 393

precipitants ( ) after SGF treatment on the adhesion of H. pylori to the gastriccarcinoma cell line AGS.

SGF for 60 min, the percentage of attached H. pylori was increasedto 88%. As the SGF exposure time was increased, the anti-adhesiveactivity of non-encapsulated 393 precipitants was decreased, lead-ing to the adherence of H. pylori to AGS cells.

The properties of H. pylori attached to AGS cells were reducedafter the exposure time of encapsulated 393 precipitants to SGF wasincreased from 0 min to 60 min. After encapsulated 393 precipitantswere treated with SGF for 60 min, the percentage of attached H.pylori cells was decreased to 32%. This is due to the gradual releaseof 393 precipitants from microspheres over 60 min. This resultalso showed that encapsulated 393 precipitants were protectedagainst acidic conditions by the CS-�-PGA matrix. Therefore, theanti-adhesive activity of 393 precipitants was maintained againstnon-encapsulated 393 precipitants.

4. Conclusions

Microspheres were formed by the electrostatic interactionbetween chitosan and poly-�-glutamic acid encapsulated 393precipitants produced by L. casei ATCC. CS-�-PGA microspheressuccessfully protected 393 precipitants against acidic conditionsand improved anti-H. pylori activity, including growth inhibitioneffects and anti-adhesive activity. Therefore, CS-�-PGA micro-spheres could be a possible delivery system for the effectiveeradication of H. pylori.

Acknowledgements

This study was supported by a Grant of the Korea Health 21R&D Project, Ministry of Health and Welfare, Republic of Korea(A050376).

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