research article development of bioadhesive...

Research ArticleDevelopment of Bioadhesive Microspheres for OralBioavailability Enhancement of Berberine Hydrochloride

Ye Zhang1 and Hongming Liu2

1Department of Pharmaceutical Sciences Zibo Vocational Institute Zibo Shandong 255314 China2Zibo Institute for Food and Drug Control Zibo Shandong 255086 China

Correspondence should be addressed to Ye Zhang yezhangye2006126com

Received 22 May 2016 Accepted 20 September 2016

Academic Editor Ruilong Sheng

Copyright copy 2016 Y Zhang and H LiuThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The objective of this study was to innovatively prepare chitosan-coated alginategelatin BBH loaded microspheres and evaluatetheir pharmaceutical characteristics and pharmacokinetics The bioadhesive microspheres were prepared using an emulsificationtechnique Three batches of microspheres were formed and their stability was evaluated BBH loaded microspheres were almostspherical with shallow elevation on surfaces The mean particle size of microspheres was 3682 120583m drug loading was 359 plusmn 001and in situ bioadhesion percentage was 9123 plusmn 82 and they achieved a sustained release with 7129 for 8 hours in vitroPharmacokinetic studies in rats indicated that the bioavailability of BBH microspheres was enhanced about 15-fold as comparedwith commercial tablets BBH microspheres exhibited a sustained-release profile over 48 h Thus chitosan-coated alginategelatinBBH loadedmicrospheres which combined the advantages of alginategelatinmicrospheres and chitosanmay be used as a sustaineddelivery system for BBH to treat duodenal and benign gastric ulcers

1 Introduction

Berberine hydrochloride (BBH) an active isoquinolinealkaloid is widely present in various traditional Chinesemedicines such as Hydrastis canadensis (goldenseal) Coptischinensis (Coptis or golden thread)Berberis aquifolium (Ore-gon grape) Berberis vulgaris (barberry) and Berberis aristata(tree turmeric) BBH is commercially used to treat duodenaland benign gastric ulcers caused by bacteria BBH has beenfound to be effective for diabetes and obesity partly viastimulating AMP-activated protein kinase activity [1 2] BBHhas also shown antidepressant activity by modulating brainbiogenic amines (norepinephrine serotonin and dopamine)oxide pathway andor sigma receptors [3ndash5] Recently BBHhas been reported as a novel cholesterol-lowering agentand it functions through a unique mechanism distinct fromstatins [6] However BBH one of the poorly water-solubledrugs and substrate of P-glycoprotein [7 8] has low mucosalpermeability [9] resulting in limited absorption in the gas-trointestinal tract which seriously limits its application anddevelopment as a pharmaceutical preparation Furthermoreintramuscular and intravenous administration of BBH could

result in adverse reactions such as anaphylactic shock anddrug eruption [10] Hence a novel drug delivery system toimprove the solubility and bioavailability of BBH has drawngreat attention of researchers in pharmaceutical industry

In the last decade microspheres have prospered enor-mously because of a variety of applications such as deliveryvesicles for drugs deoxyribonucleic acids antigens proteinsand enzymes especially for controlled or sustained drug-delivering systems employing biopolymers as raw material[11ndash13] Recently in pharmaceutical industry microsphereshave drawn great attention due to their excellent efficiencyin prolonging the half-life time of drug and improvingbioavailability of drug in vivo by controlling release rateof drug from the microspheres [14ndash17] In addition simpletechniques are involved in the preparation of microspheres[18ndash21] At present a couple of biodegradable polymers suchas chitosan alginate and gelatin have been used to preparemicrospheres [22ndash24] Chitosan is a type of excellent naturalhydrophilic polysaccharide amongmany biodegradable poly-mers and it is nontoxic and shows excellent mucoadhesiveand permeation-enhancing effects across biological surfaces[25] Chitosan exhibits antacid and antiulcer effects which

Hindawi Publishing CorporationInternational Journal of Polymer ScienceVolume 2016 Article ID 4235832 7 pageshttpdxdoiorg10115520164235832

2 International Journal of Polymer Science

may prevent or weaken drug irritation in the stomach [26]Additionally chitosan is a pH-sensitive hydrogel and atlow pH value protonated amino group (minusNH

3

+) can makechitosan molecules adhere to mucosal surfaces easily Hencechitosan has great potential in oral drug delivery system totreat gastric ulceration Alginate has bioadhesive propertiesand can also be effective in protecting mucous membranesof the gastrointestinal tract In the present study a sustaineddrug delivery system of chitosan-coated alginategelatin gas-tricmucoadhesive BBH loadedmicrospheres was designed totreat duodenal and benign gastric ulcers The microsphereswere cross-linked with 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)EDC is a condensation agent between carboxyl and aminogroups to form amide bonds to immobilize the micro-spheres and it has lower cytotoxicity compared with otherchemical cross-linking agents such as glutaraldehyde andformaldehyde [27] The microspheres were characterizedusing scanning electron microscopy (SEM) particle size anddistribution analysis and in situ bioadhesion testThe in vitrodissolution rate of microspheres was measured in simulatedgastric fluid by high-performance liquid chromatography(HPLC) and the in vivo bioavailability in rats was assayedusing tandem mass spectroscopy coupled to ultraperfor-mance liquid chromatography (UPLC-MSMS)

2 Materials and Methods

21 Materials BBH chitosan gelatin EDC and NHS wereobtained from Sigma-Aldrich Co (St LouisMOUSA) BBHcommercial tablets were bought from First TeachingHospitalof Tianjin University of TCM Span 80 was purchased fromCroda International Plc (Yorkshire UK) Sodium alginatewas obtained from Bio Basic Inc (Toronto Canada) Liquidparaffin was purchased from Tianjin No 1 Chemical ReagentFactory (Tianjin China) All other chemicals were of analyt-ical grade

22 Preparation of Chitosan-Coated AlginateGelatin BBHLoaded Microspheres BBH loaded microspheres were pre-pared by water-in-oil emulsion technique as previouslyreported [28] Briefly water phase containing sodium algi-nategelatin (2 3) (50w v) and BBH (05 g) was dissolvedin 2 (vv) acetic acid aqueous solution Oil phase containingliquid paraffin and emulsifiers (Span 80 and Tween 80 4 1ratio vv) was homogenized under stirred conditions at 40∘CLater water phase was added to oil phase (1 5 v v) understirred conditions of 450 rpm at 40∘C The prepared mixturewas kept over an ice-water bath for approximately 15min andthen 20mL of isopropanol was added to the mixture withcontinuous stirring for 10min to obtain a stable emulsionsystem 4mL of cross-linking agents (EDCNHS 4 1 ww)was dissolved in 50mM of MES buffer and they were thenadded to the inverse emulsion with continuous stirring for1 h in ice-water bath followed by stirring for 4 h in roomtemperature Then 120mL of acetone was added to endthe cross-linking reaction The microspheres were collectedby filtration Washing cycles were performed with acetoneand isopropanol before redispersion in distilled water and

subsequent lyophilization The prepared microspheres wereimmersed into the 05 chitosan acetate aqueous solutionat 30∘C for 30min Finally chitosan-coated alginategelatinBBH loaded microspheres were collected and washed threetimeswith deionizedwaterThemicrosphereswere immersedin carbodiimide solution for 12 h and they were then dried at40∘CThree batches of microspheres were formed

23 Characterization of Chitosan-Coated Microspheres

231 SEM Observation of Microspheres The shape and sur-face features of microspheres were observed using SEM(Philips XL30 Netherlands) The microspheres were sus-pended in distilled water and the dispersion was droppedon glass slide and dried at ambient atmosphere The sampleswere coated with gold for SEM observation

232 Particle Size and Distribution Analysis The micro-spheres were dispersed in distilled water and the particlesize and distribution of microspheres were measured by laserdiffractometry using LS230 Coulter (Coulter Co USA)

233 Drug Entrapment Efficiency and Loading Drug entrap-ment efficiency and loading were determined by dissolvingknown amount ofmicrospheres in 50mL of simulated gastricfluid under stirring for 48 h at 37∘C Then the supernatantwas filtered and analyzed using HPLC (Waters 2695 SystemMilford USA) Drug entrapment efficiency and loading werecalculated using the following equations

Drug entrapment efficiency

=actual BBH content

theoretical BBH contenttimes 100

Drug loading = BBH contentmicrocapsules weight

times 100

(1)

234 Stability Study The microspheres were stored at 60 plusmn2∘C 925 plusmn 5RH and 4500 plusmn 500 lx for 10 days withoutpackage Samples were withdrawn at different time andevaluated for appearance and BBH content

235 Bioadhesion of Chitosan-Coated BBH Loaded Micro-spheres The bioadhesion of chitosan-coated BBH loadedmicrospheres was investigated according to the method pre-viously reported by Rao and Buri [29] Male Sprague-Dawleyrats (weighing 450ndash500 g) were maintained at standard con-ditions and fasted overnight with free access to water untilexperiment All procedures were performed in accordancewith the National Institutes of Health Guide for the Careand Use of Laboratory Animals The experimental protocolwas approved by the Committee on Animal Research of ZiboInstitute for Food and Drug Control

The rats were anaesthetised with 5 chloral hydrate(250mgkg ip) Stomach was dissected and rinsed withphysiological saline till the mucosa was clean The tissue wasused within 2 h after dissection Then the stomach was cutlongitudinally spread and placed on amicroscope slid 50mg

International Journal of Polymer Science 3

of uncoated or coated microspheres was placed uniformly onthe mucosa of the stomach The microscope slid was thenplaced in a desiccator maintained at gt80 relative humidityand room temperature After 20min the microscope slidwas fixed in a washing jar at an angle of 45∘ The stomachmucosa was washed for 5min with 30mL of flushing liquid(09NaCl 01molLHCl pH 12plusmn01) at the rate of 01mLsusing a peristaltic pump The washings were collected driedand weighed The bioadhesion of stomach was calculatedaccording to the following equation

Bioadhesion () = 119872 minus (119866 minus 119898)119872

times 100 (2)

where M is the quality of microspheres (50mg) G is thequality of the dried residue and m is the quality of the solidin the 30mL of flushing liquid

24 In Vitro Release Studies The dissolution study was per-formed using rotating basket method (Sotax AT7 BaselSwitzerland) The microspheres and tablets equivalent to10mg BBH were dispersed in 200mL of simulated gastricfluid and stirred at 50 rpm at 37 plusmn 05∘C A 05mL ofthe suspension was withdrawn at specified time intervalsfiltered and the concentration of the supernatant was ana-lyzed using HPLC 5 120583L of the collected supernatant wasinjected into a chromatograph (Waters 2695 USA) equippedwith a UV detector (Waters 2487) and a reversed phaseProntoSIL 120-5-C

18-ace-APS column (250mm times 46mm

5 120583m Bischoff Chromatography Germany) The mobilephase was acetonitrile-water (30 70) containing 01 ofphosphoric acid and 005 of triethylamine The flow ratewas 10mLmin and the UV detector was set at a wavelengthof 263 nm

25 In Vivo Availability Studies The bioavailability of mic-rospheres was compared with BBH commercial tabletsMale Sprague-Dawley rats (weighing 200ndash250 g) were ran-domly divided into two groups of twelve rats each Therats were fasted overnight with free access to water untiladministered with microspheres (prepared by dispersing themicrospheres in distilled water) or BBH commercial tablets(24mgBBHkg)

05mL of blood samples was collected from the retinalvenous plexus into heparinized Eppendorf tubes at 0083025 050 075 1 2 3 4 6 8 12 and 24 h Blood samples wereimmediately centrifuged at 5000 rpm for 10min and stored atminus20∘C until UPLC-MSMS analysis

Tetrahydropalmatine was used as an internal standardat a final concentration of 2 ngmL 200120583L of acetonitrile-methanol (50 50 vv) solution was added to 50 120583L of ratplasma After vortex mixing for 1min the resultant mixturewas centrifuged at 15000 rpm for 10min The supernatantwas transferred to a clean tube and 10 120583L of supernatant wasinjected into a Shimadzu LC-20AD system equipped with abinary pump a micro vacuum degasser and an autoinjectorThe separation was performed on an Agilent ZORBAXXDB-C18 column (21 times 50mm 35 120583m) The mobile phaseconsisted of 01 (vv) formic acid inwater (A) and 01 (vv)formic acid in methanol The flow gradient was as follows

0ndash06min 90A 06ndash12min 90ndash2A 12ndash30min 2A30ndash31min 2ndash90A and 31ndash45min 90A at a flow rateof 045mLmin

The abovementioned UPLC system was connected withan API 4000 QTRAP mass spectrometry system via anESI interface The ESI-MS spectra of samples and referencecompounds were acquired in positive ionization modesThe set parameters were as follows the multiple reactionmonitoring mode at mz 33610 rarr 29210 (BBH) and mz3563 rarr 19210 (tetrahydropalmatine) curtain gas at 20 psiion spray voltage at 50 kV temperature at 500∘C ion sourcegas1 at 55 psi and ion source gas 2 at 55 psi

The pharmacokinetic parameters maximumplasma con-centration (119862max) and time to reach 119862max (119879max) wereobtained directly from the plasma concentration-time dataThe area under the plasma concentration-time curve (AUC)was calculated using the trapezoidal rule The values of 119862maxandAUCwere analyzed statistically using analysis of varianceafter logarithmic transformation A 119875 value lt 005 wasconsidered statistically significantThe relative bioavailabilityof microspheres was calculated using the following equationFr = AUCmicrophereAUCreference(commercial tablets)

3 Results

SEM photographs showed that particles of alginategelatinBBH loaded microspheres with deep elevation on surfaceswere spherical on the whole (Figure 1(a)) However aftercoating with chitosan the microcapsules were almost spher-ical with shallow elevation on surfaces (Figure 1(b)) Thismorphological characterization may be due to ice crystalsublimation in the process of lyophilization leading to poroussponginess-like surfaceThis could ensure microspheres withgood fluidity and large specific surface leading to improveddrug absorption Particle distribution of BBH microspheresis shown in Figure 2 The median diameter before and aftercoating with chitosan was 3020 120583m and 3682 120583m respec-tively This increase of 662 120583m was considered to be due tothe thickness of the coating layer These two microspheresdisplayed Gaussian distribution The entrapment efficiencyof BBH microcapsules was found to be 9892 plusmn 003with a drug loading of 359 plusmn 001 Bioadhesion percentageof coated microspheres was 9123 plusmn 82 while that ofuncoated microspheres was only 213 plusmn 57 The stabilityresults demonstrated that there was no significant change inthe appearance and BBH content after 10 days of exposure to60∘C 925 RH and 4500 lx

In vitro release behaviors of BBH from chitosan-coatedalginategelatin microspheres and commercial tablets areshown in Figure 3 In simulated gastric fluid about 1762of the drug loaded onto the microspheres was released in05 h After 8 hours the drug release ratio reached 7129Theinitial burst release was not apparent which was related tocross-linked microspheres coated with chitosan The releaseof BBH was faster in commercial tablets compared to BBHmicrospheres About 712 of the drug was released in 10minand 854 of the drug was released in 05 h followed by aconstant release rate


Figure 1 Scanning electron microscopy photographs of (a) alginategelatin BBH loaded microspheres and (b) chitosan-coated algi-nategelatin BBH loaded microspheres

001

20

10

01000010001001001

Particle size (120583m)

Volu

me (

)

Volu

me (

)

100

90

80

70

60

50

40

30

20

10

0

(a)

20

10

0001 1000010001001001


Volu

me (

)

Volu

me (

)

100

90

80

70

60

50

40

30

20

10

0

(b)

Figure 2 Size distribution of (a) alginategelatin BBH loaded microspheres and (b) chitosan-coated alginategelatin BBH loaded micros-pheres

0 100 200 300 400 500 600

0

20

40

60

80

100

BBH

cum

ulat

ed re

leas

e (

)

Time (min)

Figure 3 In vitro release testing of different BBH preparations at37∘C in simulated gastric fluid The preparations tested were BBHcommercial tablets (-◼-) and chitosan-coated alginategelatin BBHloaded microspheres (-e-)

The LC-MSMSmethod for the determination and quan-titation of BBH in rat plasma was developed and validatedThemean recovery was from 1021 to 1041 for 05 50 and40 ngmL of quality control samples with relative standarddeviation (RSD) of less than 5 The intra- and interdayprecisions were within 30 and 35 respectively with

accuracy from 991 to 1031 The RSD of repeatability wasless than 41 The calibration curve was 119884 = 0341119883 +000741 (119903 = 09995 119899 = 6) The linear range forthe determination of BBH was 02ndash50 ngmL and limit ofdetection (SN gt 3) was 02 ngmL

The plasma concentration-time profiles of BBH micro-spheres and commercial tablets are shown in Figure 4 Whencommercial tablets were given BBHwas undetectable at 24 hBBHmicrospheres exhibited a sustained release of BBH over48 h postfeeding The drug from microspheres was elimi-nated slowly compared to that from commercial tablets Thepharmacokinetic parameters 119862max 119879max and AUC valueswere 65 plusmn 07 ngmL 2 plusmn 046 h and 1094 plusmn 08 ngsdothmLrespectively for BBH microspheres and 77 plusmn 02 ngmL 1 plusmn037 h and 433 plusmn 03 ngsdothmL respectively for commercialtablets 119862max and 119879max of BBH microspheres were smallerand higher than commercial tablets respectively The AUCof BBHmicrospheres was higher than the commercial tabletsby 1525 and the bioavailability of BBH microspheres was252 times as much as the commercial tablets Hence BBHmicrospheres might be a promising sustained-release systemfor the oral delivery of BBH

4 Discussion

In the present study water-in-oil emulsion technique wasused to prepare BBH loaded microspheres In microsphere


0 2 4 6 8 10 12 24 36 480

1

2

3

4

5

6

7

8

9Pl

asm

a con

cent

ratio

n of

ber

berin

e (ng

mL)

Time (h)

Figure 4 Plasma concentration profiles of berberine hydrochloride(BBH) after oral administration of chitosan-coated alginategelatinBBH loaded microspheres (-◼-) and BBH commercial tablets (-e-)in rats (119899 = 4 and 24mgkg)

preparation WO ratio is a key parameter to be consideredto obtain good quality microspheres [30] The WO ratio ofhigher than 101 results in excessive hydrogelation whereasWO ratio less than 110 induces adhesion microspheres Theclaimed optimal ratio was between 225 and 25 Tominimizethe burst effect of BBH it would have been preferable touse high WO ratio according to a reported literature [31]However a high WO ratio often results in nonsphericalparticles highly variable in size and large particle size Thusa relatively high WO ratio of 15 was selected in this studyEmulsifiers also play an important role in the high-qualitymicrosphere preparation [32] Hence the influence of thetype of emulsifier was evaluated with sodium desoxycholatepolyvinyl alcohol Tween 80 and Span 80 Mixed emulsifierof Tween 80 and Span 80 produced less heterogeneous andless disrupt microspheres This could be due to the variationin the hydrophilic-lipophilic balance as well as the nature ofthe surfactant

So far many chitosan-coated particles have been reportedfor oral drug delivery The first improvement of theencapsulation process over our previous technique [33]was the enhancement of reproducibility which remains amajor issue for microsphere manufacturing process [34]To obtain reproducible batches the emulsion was madein a temperature-controlled apparatus as it was very clearthat emulsion temperature was a key process parameterdetermining the overall quality of the microspheres and thusthe release patterns [35]Hence in the early stage of emulsionhigh temperature was used to aid in the uniform dispersionof aqueous phase which thus accelerates preparation ofemulsion system Later the emulsion system was transferredto an ice-water bath to enhance shrinking droplets and toreduce the probability of collision and integration of dispersaldroplets Meanwhile it also reduces the solubility of alginateand gelatin and thus improves the gelation of alginate andgelatin In view of thermodynamics low temperature wasdisadvantageous to obtain long-term stable emulsion system

Hence dispersant agent isopropanol was added to the emul-sion system to further promote the gelation and increase thedispersibility of emulsion system Then cross-linking agentwas added to solidify the microspheres Another strategyto obtain reproducible batches was the vigorous stirring ofthe emulsion at 400 rpm which allowed a homogeneousemulsion and thus high-quality microsphere batches Thesecondmajor improvement of preparedmicrosphereswas thesurface with deep elevation which could produce larger inter-facial surface area than smooth surface and thus significantlyenhance absorption

Thebioadhesion percentage of coated BBH loadedmicro-spheres was higher than that of uncoated BBH loaded micro-spheres This indicated that coated microspheres showedbetter bioadhesion properties than uncoated microspheresUncoated microspheres were entrapped in mucosa primarilybecause of their irregular shape unlike the spherical glassbeads

The release behaviors of BBH from chitosan-coated algi-nategelatin microspheres suggested that the drug moleculeswere trapped inside the cross-linked shells and only 176of BBH was released within 30min If it was a case ofrelease from the surfacemost of the adsorbed drugmoleculescould be released within 10min when the microspheres cameinto contact with the release medium [13 36] To restrictthe burst effect several methods were used First BBH waspurified by recrystallization and then crushed into powderbecause optimized milling process could have significanteffect on the release profile [37] in particular on the bursteffect Second drug loading method was optimized as itcould influence the burst effect [30] Indeed if saturatedaqueous solution of BBHwas used as the aqueous phase drugloading of microspheres might reach up to 912 Howeverthemicrospheres showed a significant burst effectThis couldbe related to a percolation mechanism as previously reportedfor microparticles loaded with hydrophilic crystals such ascisplatin at sufficient amounts (gt18ww) [38]

The pharmacokinetic parameters suggested that thebioavailability of BBHwas significantly increased in chitosan-coated alginategelatin microspheres as compared to com-mercial tablets The results demonstrated that chitosan-coated alginategelatin BBH loaded microspheres had abetter sustained-release profile and better promoted oralabsorption of BBH than commercial tabletsThis is attributedto the bioadhesive property of chitosan and the advantagesof microspheres Thus we conclude that a chitosan-coatedalginategelatin delivery system that is a carrier combiningalginategelatin microspheres and chitosan is a favorableoption for oral administration of BBH

5 Conclusions

Spherical and uniform-sized chitosan-coated alginategelatinmicrospheres with a mean diameter of 3682120583m drug load-ing of 359 plusmn 001 and in situ bioadhesion percentageof 9123 plusmn 82 can be prepared using WO emulsionmethod In vitro dissolution experiments revealed that thedissolution of BBH from microspheres was slower thancommercial tablets and microspheres slowly released the


BBH over 8 h duration Meanwhile in vivo studies revealedthat the bioavailability of BBH microspheres as comparedto commercial tablets was 2525 Cumulatively the resultssuggested that BBHmicrospheres could be used as a possiblealternative to traditional oral formulations of BBH to improveits bioavailability

Competing Interests

The authors declare that they have no competing interests

References

[1] Y S Lee W S Kim K H Kim et al ldquoBerberine a naturalplant product activates AMP-activated protein kinase withbeneficial metabolic effects in diabetic and insulin-resistantstatesrdquo Diabetes vol 55 no 8 pp 2256ndash2264 2006

[2] N Shen X Li T Zhou et al ldquoShensong Yangxin Capsule pre-vents diabetic myocardial fibrosis by inhibiting TGF-1205731Smadsignalingrdquo Journal of Ethnopharmacology vol 157 pp 161ndash1702014

[3] S K Kulkarni and A Dhir ldquoOn the mechanism of antide-pressant-like action of berberine chloriderdquo European Journal ofPharmacology vol 589 no 1ndash3 pp 163ndash172 2008

[4] S K Kulkarni andADhir ldquoPossible involvement of L-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)signaling pathway in the antidepressant activity of berberinechloriderdquo European Journal of Pharmacology vol 569 no 1-2pp 77ndash83 2007

[5] W-H Peng K-L Lo Y-H Lee T-H Hung and Y-C LinldquoBerberine produces antidepressant-like effects in the forcedswim test and in the tail suspension test in micerdquo Life Sciencesvol 81 no 11 pp 933ndash938 2007

[6] W Kong JWei P Abidi et al ldquoBerberine is a novel cholesterol-lowering drug working through a unique mechanism distinctfrom statinsrdquo Nature Medicine vol 10 no 12 pp 1344ndash13512004

[7] G-Y Pan G-J Wang X-D Liu J P Fawcett and Y-Y XieldquoThe involvement of P-glycoprotein in berberine absorptionrdquoPharmacology amp Toxicology vol 91 no 4 pp 193ndash197 2002

[8] H-J Maeng H-J Yoo I-W Kim I-S Song S-J Chung andC-K Shim ldquoP-glycoprotein-mediated transport of berberineacross Caco-2 cell monolayersrdquo Journal of PharmaceuticalSciences vol 91 no 12 pp 2614ndash2621 2002

[9] Y Zhang Y-L Cui L-N Gao and H-L Jiang ldquoEffectsof 120573-cyclodextrin on the intestinal absorption of berberinehydrochloride a P-glycoprotein substraterdquo International Jour-nal of Biological Macromolecules vol 59 pp 363ndash371 2013

[10] J-X Zhu D Tang L Feng et al ldquoDevelopment of self-microemulsifying drug delivery system for oral bioavailabilityenhancement of berberine hydrochloriderdquo Drug Developmentand Industrial Pharmacy vol 39 no 3 pp 499ndash506 2013

[11] N Foster and B H Hirst ldquoExploiting receptor biology for oralvaccination with biodegradable particulatesrdquo Advanced DrugDelivery Reviews vol 57 no 3 pp 431ndash450 2005

[12] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1ndash3 pp 81ndash96 2001

[13] X Peng L Zhang and J F Kennedy ldquoRelease behavior ofmicrospheres from cross-linked N-methylated chitosan encap-sulated ofloxacinrdquo Carbohydrate Polymers vol 65 no 3 pp288ndash295 2006

[14] L-Y Wang Y-H Gu Q-Z Zhou G-H Ma Y-H Wanand Z-G Su ldquoPreparation and characterization of uniform-sized chitosan microspheres containing insulin by membraneemulsification and a two-step solidification processrdquo Colloidsand Surfaces B Biointerfaces vol 50 no 2 pp 126ndash135 2006

[15] M N Uddin N J Patel T Bhowmik et al ldquoEnhancedbioavailability of orally administered antisense oligonucleotideto nuclear factor kappa BmRNA after microencapsulation withalbuminrdquo Journal of Drug Targeting vol 21 no 5 pp 450ndash4572013

[16] L-D Hu Q-B Xing C Shang et al ldquoPreparation of rosigli-tazone maleate sustained-release floating microspheres forimproved bioavailabilityrdquo Pharmazie vol 65 no 7 pp 477ndash4802010

[17] M D Blanco M V Bernardo R L Sastre R Olmo E Munizand J M Teijon ldquoPreparation of bupivacaine-loaded poly(120576-caprolactone) microspheres by spray drying drug release stud-ies and biocompatibilityrdquo European Journal of Pharmaceuticsand Biopharmaceutics vol 55 no 2 pp 229ndash236 2003

[18] R K Jha S Tiwari and B Mishra ldquoBioadhesive microspheresfor bioavailability enhancement of raloxifene hydrochlorideformulation and pharmacokinetic evaluationrdquo AAPS Pharm-SciTech vol 12 no 2 pp 650ndash657 2011

[19] A Gifani M Taghizadeh A A Seifkordi and M ArdjmandldquoPreparation and investigation the release behaviour of waxmicrospheres loaded with salicylic acidrdquo Journal of Microencap-sulation vol 26 no 6 pp 485ndash492 2009

[20] S G Gayakwad N K Bejugam N Akhavein N A UddinC E Oettinger and M J DrsquoSouza ldquoFormulation and in vitrocharacterization of spray-dried antisense oligonucleotide toNF-kappaB encapsulated albumin microspheresrdquo Journal ofMicroencapsulation vol 26 no 8 pp 692ndash700 2009

[21] F Yang F-L Song Y-F Pan et al ldquoPreparation and charac-teristics of interferon-alpha poly(lactic-co-glycolic acid) micro-spheresrdquo Journal of Microencapsulation vol 27 no 2 pp 133ndash141 2010

[22] S S Xu J Wu and W Jiang ldquoSynthesis and characterisation ofa pH-sensitive magnetic nanocomposite for controlled deliveryof doxorubicinrdquo Journal ofMicroencapsulation vol 32 no 6 pp533ndash537 2015

[23] E Basaran E Yenilmez M S Berkman G Buyukkorogluand Y Yazan ldquoChitosan nanoparticles for ocular delivery ofcyclosporine Ardquo Journal of Microencapsulation vol 31 no 1 pp49ndash57 2014

[24] N Yuksel A Aydmli Y Ozalp and N Ozdemir ldquoEvaluationand preparation of controlled release lipid micropheres ofsulphamethizole by a congealable disperse phase encapsulationmethodrdquo Acta Poloniae PharmaceuticamdashDrug Research vol 57no 3 pp 187ndash192 2000

[25] T Chandy and C P Sharma ldquoChitosanmdashas a biomaterialrdquoBiomaterials Artificial Cells and Artificial Organs vol 18 no1 pp 1ndash24 1990

[26] W-M Hou S Miyazaki M Takada and T Komai ldquoSustainedrelease of indomethacin from chitosan granulesrdquo Chemical ampPharmaceutical Bulletin vol 33 no 9 pp 3986ndash3992 1985

[27] H-W Sung D-M Huang W-H Chang R-N Huang and J-C Hsu ldquoEvaluation of gelatin hydrogel crosslinkedwith various


crosslinking agents as bioadhesives in vitro studyrdquo Journal ofBiomedical Materials Research vol 46 no 4 pp 520ndash530 1999

[28] X Z Shu and K J Zhu ldquoA novel approach to preparetripolyphosphatechitosan complex beads for controlled releasedrug deliveryrdquo International Journal of Pharmaceutics vol 201no 1 pp 51ndash58 2000

[29] K V R Rao and P Buri ldquoA novel in situ method to test poly-mers and coated microparticles for bioadhesionrdquo InternationalJournal of Pharmaceutics vol 52 no 3 pp 265ndash270 1989

[30] F Lagarce P Renaud N Faisant et al ldquoBaclofen-loaded micro-spheres preparation and efficacy testing in a new rabbit modelrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol59 no 3 pp 449ndash459 2005

[31] F T Meng G H Ma W Qiu and Z G Su ldquoWOW doubleemulsion technique using ethyl acetate as organic solventeffects of its diffusion rate on the characteristics of micropar-ticlesrdquo Journal of Controlled Release vol 91 no 3 pp 407ndash4162003

[32] H Jeffery S S Davis and D T OrsquoHagan ldquoThe preparation andcharacterization of poly(lactide-co-glycolide) microparticlesIIThe entrapment of a model protein using a (water-in-oil)-in-water emulsion solvent evaporation techniquerdquo PharmaceuticalResearch vol 10 no 3 pp 362ndash368 1993

[33] Q-S Wang Y-L Cui Y Zhang Y-B Zhang and X-MGao ldquoPreparation and evaluation of chitosan-coated alginategelatin sustained releasing microspheres containing berberinehydrochloride in vitrordquo in Proceedings of the 3rd Interna-tional Conference on Bioinformatics and Biomedical Engineering(iCBBE rsquo09) pp 1ndash4 IEEE Beijing China June 2009

[34] J Herrmann and R Bodmeier ldquoBiodegradable somatostatinacetate containing microspheres prepared by various aqueousand non-aqueous solvent evaporationmethodsrdquoEuropean Jour-nal of Pharmaceutics and Biopharmaceutics vol 45 no 1 pp75ndash82 1998

[35] Y-Y Yang H-H Chia and T-S Chung ldquoEffect of prepara-tion temperature on the characteristics and release profiles ofPLGA microspheres containing protein fabricated by double-emulsion solvent extractionevaporation methodrdquo Journal ofControlled Release vol 69 no 1 pp 81ndash96 2000

[36] P He S S Davis and L Illum ldquoChitosan microspheres pre-pared by spray dryingrdquo International Journal of Pharmaceuticsvol 187 no 1 pp 53ndash65 1999

[37] A Geze M C Venier-Julienne D Mathieu R Filmon RPhan-Tan-Luu and J P Benoit ldquoDevelopment of 5-iodo-21015840-deoxyuridinemilling process to reduce initial burst release fromPLGA microparticlesrdquo International Journal of Pharmaceuticsvol 178 no 2 pp 257ndash268 1999

[38] G SpenlehauerM Vert J-P Benoıt F Chabot andMVeillardldquoBiodegradable cisplatiimmicrospheres prepared by the solventevaporation method morphology and release characteristicsrdquoJournal of Controlled Release vol 7 no 3 pp 217ndash229 1988

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


may prevent or weaken drug irritation in the stomach [26]Additionally chitosan is a pH-sensitive hydrogel and atlow pH value protonated amino group (minusNH

3

+) can makechitosan molecules adhere to mucosal surfaces easily Hencechitosan has great potential in oral drug delivery system totreat gastric ulceration Alginate has bioadhesive propertiesand can also be effective in protecting mucous membranesof the gastrointestinal tract In the present study a sustaineddrug delivery system of chitosan-coated alginategelatin gas-tricmucoadhesive BBH loadedmicrospheres was designed totreat duodenal and benign gastric ulcers The microsphereswere cross-linked with 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)EDC is a condensation agent between carboxyl and aminogroups to form amide bonds to immobilize the micro-spheres and it has lower cytotoxicity compared with otherchemical cross-linking agents such as glutaraldehyde andformaldehyde [27] The microspheres were characterizedusing scanning electron microscopy (SEM) particle size anddistribution analysis and in situ bioadhesion testThe in vitrodissolution rate of microspheres was measured in simulatedgastric fluid by high-performance liquid chromatography(HPLC) and the in vivo bioavailability in rats was assayedusing tandem mass spectroscopy coupled to ultraperfor-mance liquid chromatography (UPLC-MSMS)

2 Materials and Methods

21 Materials BBH chitosan gelatin EDC and NHS wereobtained from Sigma-Aldrich Co (St LouisMOUSA) BBHcommercial tablets were bought from First TeachingHospitalof Tianjin University of TCM Span 80 was purchased fromCroda International Plc (Yorkshire UK) Sodium alginatewas obtained from Bio Basic Inc (Toronto Canada) Liquidparaffin was purchased from Tianjin No 1 Chemical ReagentFactory (Tianjin China) All other chemicals were of analyt-ical grade

22 Preparation of Chitosan-Coated AlginateGelatin BBHLoaded Microspheres BBH loaded microspheres were pre-pared by water-in-oil emulsion technique as previouslyreported [28] Briefly water phase containing sodium algi-nategelatin (2 3) (50w v) and BBH (05 g) was dissolvedin 2 (vv) acetic acid aqueous solution Oil phase containingliquid paraffin and emulsifiers (Span 80 and Tween 80 4 1ratio vv) was homogenized under stirred conditions at 40∘CLater water phase was added to oil phase (1 5 v v) understirred conditions of 450 rpm at 40∘C The prepared mixturewas kept over an ice-water bath for approximately 15min andthen 20mL of isopropanol was added to the mixture withcontinuous stirring for 10min to obtain a stable emulsionsystem 4mL of cross-linking agents (EDCNHS 4 1 ww)was dissolved in 50mM of MES buffer and they were thenadded to the inverse emulsion with continuous stirring for1 h in ice-water bath followed by stirring for 4 h in roomtemperature Then 120mL of acetone was added to endthe cross-linking reaction The microspheres were collectedby filtration Washing cycles were performed with acetoneand isopropanol before redispersion in distilled water and

subsequent lyophilization The prepared microspheres wereimmersed into the 05 chitosan acetate aqueous solutionat 30∘C for 30min Finally chitosan-coated alginategelatinBBH loaded microspheres were collected and washed threetimeswith deionizedwaterThemicrosphereswere immersedin carbodiimide solution for 12 h and they were then dried at40∘CThree batches of microspheres were formed

23 Characterization of Chitosan-Coated Microspheres

231 SEM Observation of Microspheres The shape and sur-face features of microspheres were observed using SEM(Philips XL30 Netherlands) The microspheres were sus-pended in distilled water and the dispersion was droppedon glass slide and dried at ambient atmosphere The sampleswere coated with gold for SEM observation

232 Particle Size and Distribution Analysis The micro-spheres were dispersed in distilled water and the particlesize and distribution of microspheres were measured by laserdiffractometry using LS230 Coulter (Coulter Co USA)

233 Drug Entrapment Efficiency and Loading Drug entrap-ment efficiency and loading were determined by dissolvingknown amount ofmicrospheres in 50mL of simulated gastricfluid under stirring for 48 h at 37∘C Then the supernatantwas filtered and analyzed using HPLC (Waters 2695 SystemMilford USA) Drug entrapment efficiency and loading werecalculated using the following equations

Drug entrapment efficiency

=actual BBH content

theoretical BBH contenttimes 100

Drug loading = BBH contentmicrocapsules weight

times 100

(1)

234 Stability Study The microspheres were stored at 60 plusmn2∘C 925 plusmn 5RH and 4500 plusmn 500 lx for 10 days withoutpackage Samples were withdrawn at different time andevaluated for appearance and BBH content

235 Bioadhesion of Chitosan-Coated BBH Loaded Micro-spheres The bioadhesion of chitosan-coated BBH loadedmicrospheres was investigated according to the method pre-viously reported by Rao and Buri [29] Male Sprague-Dawleyrats (weighing 450ndash500 g) were maintained at standard con-ditions and fasted overnight with free access to water untilexperiment All procedures were performed in accordancewith the National Institutes of Health Guide for the Careand Use of Laboratory Animals The experimental protocolwas approved by the Committee on Animal Research of ZiboInstitute for Food and Drug Control

The rats were anaesthetised with 5 chloral hydrate(250mgkg ip) Stomach was dissected and rinsed withphysiological saline till the mucosa was clean The tissue wasused within 2 h after dissection Then the stomach was cutlongitudinally spread and placed on amicroscope slid 50mg




times 100 (2)











3 Results





001

20

10

01000010001001001


Volu

me (

)

Volu

me (

)

100

90

80

70

60

50

40

30

20

10

0

(a)

20

10

0001 1000010001001001


Volu

me (

)

Volu

me (

)

100

90

80

70

60

50

40

30

20

10

0

(b)


0 100 200 300 400 500 600

0

20

40

60

80

100

BBH

cum

ulat

ed re

leas

e (

)

Time (min)





4 Discussion



0 2 4 6 8 10 12 24 36 480

1

2

3

4

5

6

7

8

9Pl

asm

a con

cent

ratio

n of

ber

berin

e (ng

mL)

Time (h)








5 Conclusions




Competing Interests


References
















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






times 100 (2)











3 Results





001

20

10

01000010001001001


Volu

me (

)

Volu

me (

)

100

90

80

70

60

50

40

30

20

10

0

(a)

20

10

0001 1000010001001001


Volu

me (

)

Volu

me (

)

100

90

80

70

60

50

40

30

20

10

0

(b)


0 100 200 300 400 500 600

0

20

40

60

80

100

BBH

cum

ulat

ed re

leas

e (

)

Time (min)





4 Discussion



0 2 4 6 8 10 12 24 36 480

1

2

3

4

5

6

7

8

9Pl

asm

a con

cent

ratio

n of

ber

berin

e (ng

mL)

Time (h)








5 Conclusions




Competing Interests


References
















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





001

20

10

01000010001001001


Volu

me (

)

Volu

me (

)

100

90

80

70

60

50

40

30

20

10

0

(a)

20

10

0001 1000010001001001


Volu

me (

)

Volu

me (

)

100

90

80

70

60

50

40

30

20

10

0

(b)


0 100 200 300 400 500 600

0

20

40

60

80

100

BBH

cum

ulat

ed re

leas

e (

)

Time (min)





4 Discussion



0 2 4 6 8 10 12 24 36 480

1

2

3

4

5

6

7

8

9Pl

asm

a con

cent

ratio

n of

ber

berin

e (ng

mL)

Time (h)








5 Conclusions




Competing Interests


References
















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0 2 4 6 8 10 12 24 36 480

1

2

3

4

5

6

7

8

9Pl

asm

a con

cent

ratio

n of

ber

berin

e (ng

mL)

Time (h)








5 Conclusions




Competing Interests


References
















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





Competing Interests


References
















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



research article development of bioadhesive...

Documents