Cite this: RSC Advances, 2013, 3,19144

Chitosan coated hydroxypropyl methylcellulose-ethylcellulose shell based gastroretentive dual workingsystem to improve the bioavailability of norfloxacin3

Received 3rd June 2013,Accepted 8th August 2013

DOI: 10.1039/c3ra42726a

www.rsc.org/advances

Vivek K. Pawar,a Shalini Asthana,a Neelam Mishra,ab Mohini Chaurasia*b and ManishK. Chourasia*a

This investigation was aimed to formulate, evaluate and optimize a gastroretentive dual working system

based on mucoadhesion and floating mechanisms to improve the bioavailability of norfloxacin (NFX).

Floating microballoons (FMB) were prepared by a non-aqueous emulsification–solvent evaporation

method employing hydroxypropyl methylcellulose (HPMC) and ethylcellulose (EC) to develop a core

matrix. Furthermore, FMB were coated with chitosan by an ionotropic gelation method to impart

mucoadhesive characteristics. Prior to incorporation of NFX into mucoadhesive floating microballoons

(MFMB), a solid dispersion (SD) of NFX was prepared using a spray–freeze drying method in order to

improve the solubility of NFX. The morphological characteristics of the microballoons were assessed using

scanning electron microscopy (SEM) which revealed the spherical shape of the microballoons with a

smoother, dense and less porous surface. The developed microballoons were evaluated for various

physicochemical parameters such as particle size, surface morphology, entrapment efficiency (EE), in vitro-

dissolution, in vitro buoyancy and mucoadhesion. The optimized microballoons were developed with good

in vitro-buoyancy coupled with high EE. Microballoons exhibited a zero-order release in simulated gastric

fluid (SGF) demonstrating drug release in the range from 64.99 ¡ 3.26 to 99.94 ¡ 8.45% after 10 h

through various formulations. Chitosan coating over FMB imparted excellent mucoadhesion in rat gut wall

and results were also supported by mucin glycoprotein assay. MFMB were able to achieve higher mean

plasma concentrations compared to FMB and pure NFX in Wistar rats. Keeping in mind the

comprehensible advantages of a developed gastroretentive dual working system over a conventional

gastroretentive drug delivery system, it can be concluded that the developed system can be used to target

drugs in the gastric cavity.

A Introduction

Gastroretention of a drug delivery system is a useful tool toimprove pharmacotherapy of various drugs via targeting site-specific release in the upper gastrointestinal tract (GIT).Gastroretentive drug delivery systems (GRDDS) have thecapacity to release drugs in a controlled manner. They canimprove the bioavailability of drugs which exhibit a narrowabsorption window or those primarily used to treat localailments of the gastric region. Moreover, site-specific releasein the gastric region is highly desirable for drugs which areunstable or have low solubility at intestinal and colonic pH.1

These systems can improve the performance of the sustained

release dosage form by avoiding their undesirable transitionfrom GIT without releasing the complete active moiety.GRDDS have been formulated based on various approachesincluding low density systems having the capability to bebuoyant over the gastric fluid, high density systems exhibitingthe potential to reside in the bottom of the gastric cavity,mucoadhesive systems, swelling or unfolding systems andmagnetic systems controlled by an external magnetic field overthe stomach.2 Furthermore, these technologies are associatedwith merits and demerits because these products face variousphysiological difficulties such as the fed and unfed state of thestomach, the unpredictable nature of the gastric emptyingprocess and gastric mucus secretion.3

Dual working systems have gained increased attention overthe last few years. Basically, these systems have beenestablished on the basis of two or more gastroretentivetechniques such as floating–mucoadhesion,4,5 floating–swel-ling6,7 and floating–swelling–mucoadhesion.8 Dual workingsystems can overcome drawbacks associated with other

aPharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow, UP, India

226031. E-mail: manish_chourasia@cdri.res.in; Fax: +91 522 2623405; Tel: +91 522

2612411-18bAmity Institute of Pharmacy, Amity University, Lucknow, UP, India 226010.

E-mail: mohinichaurasia@gmail.com; Tel: 0522-2399418

3 CDRI Communication No. 8513.

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19144 | RSC Adv., 2013, 3, 19144–19153 This journal is � The Royal Society of Chemistry 2013

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gastroretentive technologies. For example, floating drugdelivery systems have a lower density in comparison to gastricfluid endowing the capability to remain buoyant in the gastriccavity. However, if the stomach is empty then the system maybe evacuated prematurely from the gastric cavity leading todose dumping or reduced buoyancy. Whereas, the perfor-mance of mucoadhesive drug delivery systems is totally basedon the mucus turnover rate in the stomach. Development of adual working system based on the floating–mucoadhesionprinciples of gastroretention would overcome these drawbacksand could significantly improve the performance of thegastroretentive system.9

Single unit floating systems (floating tablets) are lacking inperformance due to the ‘‘all-or-nothing’’ emptying processwhich ultimately extends to a high variation in bioavailabilityand also produces gastric irritation due to the large amount ofdrug release at a particular site in the GIT. On the other hand,multiparticulate systems (e.g. microballoons) have demon-strated improved performance over the single unit floatingsystems. They reduce the chances of gastric irritation andintersubject variability in absorption by uniformly distributingin the stomach.10

NFX is a second generation, synthetic, broad spectrumfluoroquinolone antibacterial agent. NFX is mainly indicatedfor urinary tract infections11 and sexually-transmitted diseasesviz. gonorrhoea caused by Neisseria gonorrhoeae (Gram-negative diplococcus).12 Sometimes it is also indicated incombination therapy of gastric ulcers caused by Helicobacterpylori.13 The major problem in oral administration of NFX islow oral bioavailability (30–40%) which is highly influenced bythe physiology of the GIT. Absorption of NFX mainly takesplace from the upper GIT whereas it is least absorbed form thelower GIT.14,15

NFX has a pH dependent solubility exhibiting poorsolubility at pH 7 (0.40 mg ml21) and pH 7.5 (0.45 mgm121). NFX exists in four protonated forms in the aqueousphase. The zwitterionic form of NFX is prominent at itsisoelectric point near pH 7 where it displays the leastsolubility. NFX is precipitated at neutral to slightly alkalinepH and hence it has low absorption in the lower GIT.16

Solubility of poorly soluble drugs can be improved bypreparation of SD17 and cyclodextrin inclusion complexes.18

Chitosan is a natural aminopolysaccharide derived fromchitin by partial deacetylation. Chitosan has excellent bio-compatibility and low toxicity. The major point of interest inthe case of chitosan is its mucoadhesive properties and filmforming capacity.19–21 Apart from chitosan, some othercellulose derivatives like HPMC and EC have also beenemployed to achieve controlled release characteristics. HPMCis a partly O-methylated and O-(2-hydroxypropylated) cellulosewhereas; EC (ethyl ether of cellulose) consists of a long chainof b-anhydroglucose units joint together with acetal linkages.These hydrophilic polymers are known for their excellentviscosity enhancing and swelling characteristics upon contactwith water or biological fluids which causes chain relaxationand volume expansion. The swellable nature of HPMC and EC

imparts floatability to the dosage form by entrapping air insidethe swelling matrix and subsequently releasing the drug in acontrolled fashion. Various studies have shown that HPMCand EC have the capability to form a floating core and cancontrol drug release.22,23

Keeping in mind all the above prospects, we have developeda gastroretentive dual working system based on the floating–mucoadhesion principles of gastroretention. The present workbasically deals with the formulation of chitosan-coatedmicroballoons bearing SD of NFX using different release-ratecontrolling polymers by a non-aqueous emulsification–solventevaporation method. The developed formulation was char-acterized for different levels by in vitro, ex vivo and in vivomethods to evaluate the performance of the gastroretentivesystem and bioavailability of NFX. On the basis of preliminarytrials, ratios of polymers and emulsifiers, and chitosanconcentration were selected as the formulation variables forthe optimization of the developed system.

B Experimental methods

B.1 Materials

NFX, Ethyl cellulose 45 cps (EC), Hydroxypropyl methylcellulose 80–120 cp (HPMC), polyethylene glycol-6000 (PEG-6000), beta-cyclodextrin (b-CD) and chitosan were purchasedfrom Sigma Aldrich, Inc., USA. Tween 80, Span 80 and liquidparaffin were purchased from Himedia Laboratories Pvt Ltd.,Mumbai, India. All other reagents were of analytical grade andwere used as received.

B.2 Preparation of SD

A modified spray–freeze drying method was used to prepareSD.24 Initially, a complex of NFX and b-CD was prepared by akneading technique. Both NFX and b-CD (1 : 1) were trituratedand dissolved in a 20 : 30 (v/v) mixture of ethanol and n-butylalcohol. An aqueous solution of PEG 6000 was prepared (0.6%)and both the aqueous and organic phases were mixed in afinal ratio of 50 : 50 to obtain a drug–carrier ratio of 1 : 2 (w/w). The above clear mixture was fed into a spray dryer (LU 20Spray Dryer, Labultima, Mumbai, India) and sprayed througha twin-fluid nozzle. Spray–freezing was performed by engaginga liquid nitrogen droplet surface and the distance betweennozzle and liquid nitrogen surface was kept constant at 4 cm.The pressure of nitrogen was adjusted to 4 bar and the flowrate of the feeding solution was set at 20 ml min21. Afterspray–freeze drying, the samples were lyophilized and storedin vacuum desiccators at 45.3 Pa for 72 h. A physical mixturewas prepared using the same drug–carrier ratio as specifiedabove.

B.3 Preparation of FMB and MFMB

A non-aqueous emulsion solvent evaporation (Oil-in-Oil)method was used with slight modifications to prepareFMB.25 Various weight ratios of EC and HPMC were dissolvedin an equal volume blend of acetone and isopropyl alcohol (10ml). NFX and its SD (NFX concentration 50 mg) wereincorporated into the above solution along with Tween 80

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(0.2%). The developed slurry was added to 20 ml of liquidparaffin containing Span 80 (0.3%) to prepare a primaryemulsion. The emulsion was stirred at 1200 rpm for 2 h atroom temperature using a 4-bladed angular stirrer (IKA, RW20, Staufen, Germany) to completely evaporate the solvents.The developed microballoons were filtered and washed withn-hexane in order to completely remove the oil and dried atroom temperature by keeping in the desiccator for 2 days.

Furthermore, FMB were coated with chitosan via anionotropic gelation method. Chitosan at different concentra-tions was dissolved in the 2% acetic acid solution (40 ml) andthe above solution was sprayed over the FMB using a spraydryer at a flow rate of 20 ml min21 and atomization pressure of4 bar. Afterwards, 1 N sodium hydroxide solution (15 ml) wassprayed over the FMB using similar atomization conditions.The developed MFMB were dried under filtered, hot andpressurized air. The MFMB were separated by the means of acyclone separator and remaining traces of solvent wereevaporated by vacuum drying. The concentration of chitosanwas optimized on the basis of the zeta potential of thedeveloped MFMB.

B.4 Determination of particle size and zeta potential

Samples for particle-size and zeta-potential determinationwere prepared by suspending the MFMB in 0.001 M phosphatebuffer (pH 7.0) by ultrasonication for 0.5 min and theconcentration of the suspension was adjusted to 2% w/v.Particle size distribution of the MFMB was determined using aMalvern MasterSizer S instrument (Malvern Instruments Ltd.,Malvern Worcestershire, UK).

Zeta potential was measured on the basis of electrophoresisusing a nanoZS Malvern Zetasizer (Malvern Instruments Ltd.,Malvern Worcestershire, UK) equipped with integral goldelectrodes in order to measure the zeta potential.

B.5 Surface and internal morphology

The surface and internal morphology were examined using ascanning electron microscope (EVO-50, ZEISS, UnitedKingdom). The samples for SEM were prepared by adheringthe microballoons on a double adhesive tape stuck to analuminium stub. These stubs were then coated with a gold–palladium alloy in a vacuum evaporator (SC7640 PolaronSputter Coater). The internal morphology of the microballoonswas assessed from the craters on the surface of themicroballoons.

B.6 Determination of entrapment efficiency

EE of MFMB was determined by extracting the NFX in amixture of water and ethanol. The MFMB (20 mg) were soakedovernight in the 20 ml water and ethanol mixture (6 : 4)followed by vortexing for 5 min. The above solution was thencentrifuged at 12 000 rpm at 5 uC for 10 min and thesupernatant was assayed for NFX content with a UV-spectro-photometer at 273 nm (UV 1700 Shimadzu, Japan).

B.7 Buoyancy test

For the determination of buoyancy, MFMB (300 mg) werespread over the surface of the 100 ml SGF, pH 1.2, containing0.2% w/v of Tween 20 and subjected to stirring at 100 rpm.

After 12 h, floating and settled MFMB were collected separatelyand both the fractions were dried and weighed to calculate thebuoyancy using eqn (1):26

% Buoyancy = [QF/QF + QS] 6 100 (1)

Where, QF and QS are the weight of the floating and thesettled microballoons, respectively.

B.8 Dissolution studies

The dissolution studies were conducted using a USP type IIapparatus. MFMB comprising NFX (22 mg) and SD of NFX (50mg) were placed in the dissolution vessel having 500 ml SGF(pH 1.2) containing Tween 20 (0.2% w/v) as the dissolutionmedium. The paddle was rotated at 100 rpm and thetemperature was maintained at 37 ¡ 0.5 uC. Aliquots (5 ml)were withdrawn at 1 h intervals up to 10 h and replaced withan equal volume of fresh medium to maintain a constant totalvolume and sink condition. Aliquots were filtered through amembrane filter (0.45 mm) and assayed for drug content afterappropriate dilutions at 273 nm using a UV spectrophotometer(UV 1700 Shimadzu, Japan). All the release studies wereconducted in triplicate. Drug release from all formulationswere statistically analysed by two-way analysis of variance(ANOVA) followed by Student t-tests. A statistically significantdifference between in vitro drug releases was defined as p ,

0.05 using the Graph pad Instat software (Graph Pad SoftwareInc. CA, USA).

B.9 Dissolution model-dependent kinetics

The dissolution data was analyzed and plotted for variousrelease kinetic models such as zero-order (eqn (2)), first-order(eqn (3)), Higuchi matrix (eqn (4)), Korsmeyer–Peppas (eqn (5))and Hixson–Crowell (eqn (6)). The linearity was calculated todetermine th best fit model and possible mechanism of drugrelease from the microballoons.27

R = k1t (2)

log UR = k2t/2.303 (3)

R = k3!t (4)

log R = log k4 + n log t (5)

UR1/3 = k5t (6)

Where,R and UR are the released and unreleased drug percentages,

respectively, at time (t);k1, k2, k3, k4, and k5 are the rate constants of zero-order, first-

order, Higuchi matrix, Korsmeyer–Peppas, and Hixon–Crowellmodel, respectively.

B.10 Stability of MFMB in 0.1N HCl

In order to determine the stability, MFMB were incubated in0.1 N HCl for 60 min at a final concentration of 0.5% w/v andthe % transmission of the samples was measured using a UV-spectrophotometer (UV 1700 Shimadzu, Japan). Basically, thistest evaluated the effect of chitosan concentration on its

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stability in acidic pH because it is well established that thechitosan is soluble at acidic pH.28

B.11 Mucous glycoprotein assay

For mucous glycoprotein assay, mucin was dissolved in 50 mlwater in order to prepare solutions of different concentrations(500 mg, 1000 mg, 1500 mg and 2000 mg). 20 mg each of FMBand MFMB were dispersed in the above solutions, vortexedand shaken at room temperature for 1 h using a water bathshaker. After adsorption, the samples were centrifuged at 4000rpm for 5 min and the content of free mucin was measured insupernatant by a calorimetric method using periodic acid/Schiff as a staining agent.29 Two-way analysis of variance(ANOVA) was performed to determine the difference in theadsorption of mucin over FMB and MFMB at differentconcentrations. All the calculations were performed usingthe Graph Graph pad Instat software (Graph Pad Software Inc.CA, USA). P , 0.05 denotes significance in all cases.

B.12 In vivo studies

B.12.1 Animal host. Wistar rats (200–250 g) were used asanimal models for the pharmacokinetics and mucoadhesionstudy. Animals were kept in standard laboratory conditions(room temperature, 22 ¡ 1 uC; a cycle of 12 h of light and 12 hof dark with lights on at 7 am) Guidelines of Council for thePurpose of Control and Supervision of Experiments onAnimals (CPCSEA) Ministry of Social Justice andEmpowerment, Government of India were followed duringthe study.

B.12.2 Mucoadhesion studies in rat gut loop. The mucoad-hesive property of the MFMB was evaluated using male Wistarrats and the animals were kept on a fasting diet for 18 h priorto commencement of the study with free access to water (adlibitum). Animals were anaesthetized and sacrificed to removethe small intestine. The dissected intestine was washed withphysiological saline (approx. 500 ml) using a syringe at 5 to 10ml min21 for 10 min followed by 20 to 30 ml min21 for 20 min.The washed intestine was afterwards kept at 215 uC untilfurther use.30 Samples were prepared for the study in a similarmanner as those prepared for the assessment of particle sizeand zeta potential except the final concentration of thesuspension was adjusted to 4%. These sealed small intestineswere incubated at 37 uC for 60 min. Subsequently, the sampleswere removed and analyzed using a Coulter counter (BeckmanCoulter Inc., CA, USA) to count the number of microballoonspresent in the sample before and after the mucoadhesionstudy.

B.12.3 pharmacokinetics study. For the pharmacokineticstudy, animals were divided into 3 groups composed of 5healthy animals. All the groups were fasted for 12 h prior and24 h following the experiment with free access to water (adlibitum). According to the study protocol, all the groupsreceived an NFX dose equivalent to 20 mg kg21 body weightof rats. Free NFX, FMB and MFMB were administered in group1, group 2 and group 3, respectively. The samples wereadministered orally with the aid of a cannula with 3–5 ml ofwater to ensure that the animal does not chew the micro-balloons during administration. Blood samples (1 ml) were

collected by cardiac puncture at 0 (predose), 1, 2, 4 and 8 hafter administration of the treatment/formulation. Sampleswere centrifuged and the supernatant was collected for thequantification of the available NFX in the serum. A RP-HPLCmethod was used with slight modification for the estimationof the NFX in the samples.31 The HPLC system composed of abinary gradient pump (LC-20AT), UV-VIS detector (SPD-10AVVP) and a system controller (SCL-10A) (Shimadzu, Kyoto,Japan). The chromatographic and the integrated data wererecorded using a CLASS-VP workstation. For the chromato-graphic separation of NFX, a C18 column (LiChrospher, 5 mm,250 mm 6 4 mm) was used with a mobile phase composed ofacetonitrile (60%) in phosphate buffer solution and the pH ofthe buffer was adjusted to 3.5 with orthophosphoric acid. Thesamples were injected at a flow rate of 1.2 ml min21 andestimation of NFX was performed at 273 nm.

C Results and discussion

C.1 Effect of HPMC : EC ratio

MFMB were successfully prepared via a non-aqueous emulsionsolvent evaporation method followed by coating of chitosanvia an ionotropic gelation method. The ratios of EC and HPMCwere optimized by evaluating their effect on particle size, % EEand % buoyancy of the developed microballoons. The polymerratio was optimized by keeping all other parameters constantsuch as Tween 80 (0.2%), Span 80 (0.3%), acetone : IPA (1 : 1)and it was found that both the polymers significantly affect theparticle size, % EE and % buoyancy of the microballoons.Table 1 shows the effect of the polymer ratio on the particlesize of the microballoons and % EE. The mean particle size ofthe microballoons was significantly increased with increasingconcentration of EC whereas, decreasing concentration ofHPMC led to an increase in the mean particle size of themicroballoons. The mean particle size of all the formulationswas found in the range of 163.4–733.5 mm. It would beexpected that the viscosity of the polymer mixture wouldincrease as the polymer concentration increases resulting inan enhanced interfacial tension which avoids particle sizereduction due to agitation and generating larger particles. Themean particle size of free NFX loaded microballoons wassmaller than that of the SD loaded microballoons. The largersize of the SD loaded microballoons is due to the enhancedviscosity of the polymeric solution caused by the presence ofPEG-6000 in the SD.

Almost opposite results were found for the % EE of themicroballoons. Maximum entrapment of 94.72 ¡ 2.31% and88.16 ¡ 1.94% were observed for the formulations NSDMB-1and NSDMB-2 containing HPMC : EC blend ratio 1 : 1 and1 : 2, respectively. Results (Table 1) showed that the EE wasdecreased with increase in the concentration of the ECwhereas, EE was found drastically increased with increase inthe concentration of the HPMC (NSDMB-6 & NSDMB-7). Thiscould be due to the hydrophilic nature of the HPMC whichultimately provides a favourable environment to incorporatethe SD (comprising other hydrophilic polymers like PEG-6000and b-CD) in the microballoons. In comparison to NFX, its SD

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was more entrapped in the microballoons when NFX and SDcarrying equivalent amounts of NFX were incorporated in themicroballoons (NSDMB3 and NMB). EE of the microballoonsis highly regulated by the relative solubility of drug/SD in thedispersed and continuous phase. High EE was found in almostall the formulations except NSDMB7. The high EE might beattributed to the low solubility of drug and SD in the oilydispersion medium.

Results revealed that the buoyancy of the developed systemwas found to be increased when both EC and HPMC were usedin combination rather than when they were used alone. EC hasa more prominent effect on the buoyancy in comparison toHPMC. The highest buoyancy of 94.5% was observed withformulation NMB entrapping NFX whereas NSDMB-3 entrap-ping SD exhibited 92.32% buoyancy but the difference wasstatistically insignificant. The decreased extent of buoyancy informulations having a higher amount of HPMC could be dueto its hydration effect that favours the formation of more poresand channels within the microballoons causing increased

water diffusion which over time led to a change in density andsubsequent settling down of the microballoons. Decreasedbuoyancy of the SD incorporated formulation might beassociated with the presence of PEG-6000 in the SD mimickingthe action of HPMC and imparting more hydration to themicroballoons.

C.2 The effect of the emulsifier ratio

To study the effect of the emulsifier ratio all other formulationvariables were kept constant and the ratio of emulsifier waschanged to observe its effect on the particle size of themicroballoons. The different ratios used and correspondingparticle size are represented in Fig. 1. It was found that if theconcentration of the surfactant was increased the particle sizeof the microballoons was decreased up to a certain limit. Theconcentration of Tween 80 has a more intense effect on theparticle size in comparison to Span 80. However, simultaneousincrements in the concentration of both the polymersdemonstrated excellent results. The decrease in particle sizeof the microballoons on increasing the concentration of theemulsifier suggested that surfactant modified the totalinterfacial area and the size of the microballoons during theformation of the primary emulsion.

Table 1 Physicochemical characterisation of various developed formulations

Batch Code NFX (mg) S.D. (mg) HPMC : EC Chitosan (%) Shape Mean particle size (mm) % Entrapment efficiencya % Buoyancy

NSDMB1 — 50 1 : 1 2.5 Spherical 497.2 94.72 ¡ 2.31 80.43NSDMB2 — 50 1 : 2 2.5 Spherical 599.0 88.16 ¡ 1.94 87.29NSDMB3 — 50 1 : 3 2.5 Spherical 658.2 83.25 ¡ 2.89 92.32NSDMB4 — 50 0 : 1 2.5 Spherical 733.5 60.23 ¡ 1.85 86.70NSDMB5 — 50 1 : 0 2.5 Spherical 163.4 68.22 ¡ 2.25 55.01NSDMB6 — 50 3 : 1 2.5 Spherical 206.9 63.26 ¡ 8.23 65.03NSDMB7 — 50 2 : 1 2.5 Spherical 293.0 38.81 ¡ 3.98 70.13NMB 50 — 1 : 3 2.5 Spherical 390.5 75.22 ¡ 8.65 94.15

a (Mean ¡ SD, n = 3).

Fig. 1 Particle size of the microballoons when different ratios of Tween 80 andSpan 80 was used. Initially, the concentration of Tween 80 was kept constant toevaluate the effect of the concentration of Span 80 on the particle size and theresults suggested that the particle size of the microballoons was decreased withincreasing concentration of Span 80. However, the particle size of themicroballoons was drastically decreased when the concentration of bothsurfactants was increased which demonstrates that Tween 80 has a moreintense effect on the particle size of the microballoons.

Fig. 2 Effect of the concentration of chitosan on the zeta potential, %transmittance and buoyancy of the microballoons. Optimization of the chitosanconcentration was performed on the basis of the observed zeta potential of themicroballoons and the optimized chitosan concentration was found on the basiswhere the zeta potential of the microballoons becomes stabilized. The graphshows that the stability of the microballoons was increased in acidic mediumwhen the concentration of chitosan was increased from 1 to 3%; suggested by a7.6 fold decrease in % transmittance. However, the chitosan concentration doesnot show any significant effect on the buoyancy of the microballoons.

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C.3 Optimization of the chitosan concentration

The developed FMB were coated with chitosan to impartmucoadhesive properties. The concentration of chitosan wasoptimized by keeping constant all the other formulation andprocess parameters. The zeta potential of the uncoatedmicroballoons was negative (214.3 ¡ 1.2) whereas that ofthe chitosan coated microballoons was observed to be positive(Fig. 2). The concentration of chitosan has a marked effect onthe zeta potential of the microballoons and was found toincrease from 8.4 ¡ 2.3 mV to 49.5 ¡ 3.8 mV when theconcentration of chitosan was altered from 1 to 2.5%. Nosignificant deviation in zeta potential was observed above the2.5% concentration of chitosan therefore this concentrationwas selected as the optimum for coating FMB.

Furthermore, the concentration of the chitosan was opti-mized by measuring % transmittance after exposing MFMB to0.1 N HCl. The study is based upon the solubility of thechitosan in the acidic environment. The turbidity of the

sample is directly proportional to the dissolution/disintegra-tion of the MFMB. A high transmittance reveals that theformulation is unstable in acidic medium whereas a lowtransmittance indicates that the microballoons are stable inthe acidic medium. The results demonstrated that %transmittance was decreased from 64.7 to 8.45 upon incre-mental increases in the concentration of chitosan form 1 to3% (Fig. 2). This decline in transmittance might be due to theenhanced viscosity of the coating solution which arisesbecause of the increase in the concentration of chitosan. Theincreased viscosity ultimately led to the development of athicker and denser coat over the microballoon which was noteasily dissolved/swelled by the HCl. The concentration ofchitosan in the coating solution has a very much less intenseeffect over the buoyancy of the MFMB which was recorded inthe range from 86.34% to 92.32%. The feeble outcome mightbe attributed to the dual effect of the chitosan concentrationon the buoyancy of the MFMB. Initially, chitosan masks theavailable pores over the surface of the FMB which is counter-

Fig. 3 SEM photomicrographs of FMB and MFMB: [A] general appearance of FMB (uncoated microballoons) [B] Surface morphology of the FMB showing numerouspores over the surface of the microballoons [C] general appearance of MFMB (chitosan coated microballoons) [D] Surface morphology of the MFMB exhibiting asmooth and less porous surface in comparison with the surface of the FMB (E) View of the internal morphology of the MFMB; showing an irregular internal surfaceformed due to evaporation of acetone.

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acted by the sinking action of water going through the poresinto the microballoons ultimately leading to increased buoy-ancy. On the other hand, subsequently it might increase thedensity of the microballoons leading to a decrease in thebuoyancy of the MFMB and neutralizing the overall effect ofthe chitosan concentration on the buoyancy.

C.4 SEM analysis

SEM photomicrographs revealed that developed FMB (Fig. 3A)and MFMB (Fig. 3C) were predominately spherical in shape.The surface of the MFMB (Fig. 3D) is smoother, dense and lessporous in comparison to the surface of the FMB (Fig. 3B). Thegeneration of a highly porous and rough surface of the FMBmight be due to incorporated SD in the shell of themicroballoons along with initial evaporation of isopropylalcohol during the formation of the microballoons.Apparently the surface of the MFMB was visibly smooth,however when the magnification of the machine was increasedin order to check the surface morphology, it was observed thatMFMB exhibited an irregular internal surface with signs ofnumerous depressions formed due to the evaporation ofacetone (Fig. 3E). This excellent configuration of the morphol-ogy of MFMB supports controlled drug release from themicroballoons along with a splendid buoyancy.

C.5 Dissolution studies

In vitro release studies were conducted in SGF pH 1.2 for 10 h.To simulate the fasting state conditions of the stomach Tween20 was added in the dissolution medium to assist de-aggregation of the microballoons. The results of the drug

release studies are presented in Fig. 4 and indicate that NFXrelease from the microballoons is strictly dependent upon theconcentration of both the polymers. The drug release from allthe formulations was found to range from 64.99 ¡ 3.26–99.94¡ 8.45%. EC was found to exhibit a dominant effect on drugrelease from the microballoons in comparison to HPMC. Thedecrease in drug release from 98.37 ¡ 3.32 to 64.99 ¡ 3.26 onraising the concentration of the EC might be attributed to itsless aqueous permeability which ultimately increases thepolymer matrix density and hence the path length to befollowed by NFX from the matrix of the polymer. On the otherhand, HPMC is a hydrophilic polymer which imparts a moreporous and less dense structure to the microballoonseventually increasing drug release from 79.49 ¡ 2.45% to92.58 ¡ 3.78% when its concentration was increased (NSDMB-6 & NSDMB-7). Controlled drug release from the microbal-loons is also attributed to the chitosan coating over themicroballoons providing a means to increase the total pathlength of the polymer matrix and mimicking the function ofEC. The release of drug from NFX loaded microballoons wasslow compared to SD loaded microballoons which is attributedto the increased solubility of NFX by PEG 6000 and b-CDavailable in the SD. On application of ANOVA, a significantdifference (P , 0.05) was recorded in the drug release profilesfrom all the formulations.

C.6 Release kinetics

Various kinetics models such as zero order, first order,Higuchi matrix, Korsmeyer–Peppas and Hixson–Crowell wereemployed to assess the release kinetics of the microballoons.Results (Table 2) suggest that the best model fit was a zeroorder-model because higher R2-values were observed com-pared to the other models for most of the formulations exceptfor NSDMB 1 and NSDMB 5. Korsmeyer–Peppas was thesecond best fit model exhibiting n values ranging from 0.45 to0.89 signifying drug release from microballoons followed bynon-Fickian or anomalous transport. The non-Fickian modelrepresents both the drug diffusion in the hydrate matrix andthe polymer relaxation phenomena for drug release. Theseresults revealed the swelling nature of the chitosan andcellulose derivatives such as HPMC and EC used in theformulation.

C.7 Mucoadhesion of MFMB

Chitosan is known to show a strong interaction with mucin.The mucin glycoprotein assay was employed to assess themucoadhesive characteristic of the microballoons. A suspen-

Fig. 4 Graph showing the effect of HPMC and EC concentration on drug releasefrom the developed microballoons. It was found that both the polymerssignificantly controlled the drug release.

Table 2 Results of kinetics model fitting for in vitro drug release data

Batch No. Zero order First order Higuchi Korsmeyer–Peppas n Hixson–Crowell

NSDMB 1 0.9753 0.9098 0.9926 0.9939 0.5735 0.9810NSDMB 2 0.9954 0.9860 0.9846 0.9827 0.4870 0.9919NSDMB 3 0.9941 0.9741 0.9669 0.9680 0.5774 0.9829NSDMB 4 0.9908 0.8590 0.9567 0.9698 0.6868 0.9205NSDMB 5 0.9317 0.8739 0.9623 0.9762 0.5043 0.9268NSDMB 6 0.9921 0.9770 0.9820 0.9804 0.4740 0.9862NSDMB 7 0.9995 0.9077 0.9860 0.9871 0.4894 0.9573NMB 0.9952 0.9774 0.9703 0.9783 0.7461 0.9857

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sion of the different formulations (FMB and MFMB) wasincubated in aqueous solution of different concentrations ofmucin. Fig. 5 shows the mucin adsorbed on the microballoonsafter incubation with mucin. It was found that the mucinadsorption over MFMB increased from 64.8 ¡ 1.34% to 84.5¡ 3.21% with increase in the amount of mucin from 10 mgml21 to 40 mg ml21. However, only a small amount of mucinwas found to be adsorbed on the FMB. The maximumadsorption of mucin on FMB was found to be 25.65 ¡

1.78%. Application of two-way ANOVA recorded a significantdifference between FMB and MFMB with respect to adsorptionof mucin (P , 0.05). The adsorption of mucin over themicroballoons is dependent on the zeta potential carried bythe formulation. MFMB exhibited a high positive zetapotential compared to negatively charged FMB which explainsthe fact that mucin adsorbed more on MFMB rather thanFMB.32

Furthermore, the mucoadhesive ability of the microballoonswas assessed by counting the unadsorbed microballoons afterincubating the coated and uncoated formulations with rat gutloop tissue. It was found that only a small amount of theuncoated microballoons were adhered to the tissue incomparison to the coated microballoons (Table 3). Theseresults further support the strong interaction of the MFMB tomucus glycoprotein and/or mucosal surfaces. The highmucoadhesive property of MFMB is mainly due to chitosanwhich is reported to have mucoadhesive properties.33,34

However, the small extent of mucoadhesion exhibited byFMB might be due to the presence of HPMC which is alsoreported to have mucoadhesive properties.35

C.8 Pharmacokinetics of NFX

The effect of different developed formulations on thebioavailability of NFX was evaluated in Wistar rats. The

formulation and free NFX was administered at a doseequivalent to 20 mg kg21 body weight. The pharmacokineticsdata of free NFX, FMB & MFMB was calculated usingWinNonlin (5.1; Pharsight, Mountain View, CA) and summar-ized in Table 4.

The serum drug concentration time profile of reference anddifferent tests is presented in Fig. 6 which shows a cleardivergence in Cmax and Tmax of the free NFX, FMB and MFMB.In the case of free NFX, absorption was quick with a slowelimination while in the cases of FMB and MFMB theabsorption was slow and prolonged. These results suggestthat the drug was slowly released from the FMB and MFMBwith subsequent absorption. In all the cases, NFX wasrecorded in the blood 8 h post administration. However, themaximum drug concentration after 8 h was found in the caseof MFMB followed by FMB and free NFX, respectively. Thismight be attributed to the elimination of already absorbeddrug and continued absorption of the controlled release NFXfrom the MFMB and FMB through GIT. The Cmax of the freeNFX, FMB and MFMB was 1.78 ¡ 0.05 mg ml21, 1.65 ¡ 0.09mg ml21 and 1.48 ¡ 0.09 mg ml21 indicating that the drugrelease was well controlled from the gastroretentive formula-tions followed by in vivo absorption of NFX.

The AUC0–‘ of NFX was increased 2.1 and 1.44 fold whenadministered through the MFMB and FMB, respectively.Moreover, the MRT was increased by 2.34 and 1.77 foldcompared to free NFX with MFMB and FMB respectively, witha corresponding decrease in clearance and elimination rateconstant. The results clearly demonstrate the improvedbioavailability of NFX. Development of a gastroretentive drugdelivery system comprising NFX is responsible for itsimproved bioavailability. Low bioavailability of native NFX isassociated with its absorption pattern in GIT. Basically, it is

Fig. 5 Graph showing the pattern of adsorption of mucin over FMB and MFMBby the mucin glycoprotein assay. A higher absorption of mucin over MFMB incomparison to FMB suggests a heightened mucoadhesive nature of developedMFMB. (P , 0.05 when comparing FMB and MFMB using the two-way ANOVAtest).

Table 3 Mucoadhesive measurements of MFMB on rat small intestine byCoulter counter measurement

Formulationcode

Counts of Microballoons

Before Incubationa After IncubationaAdhered(%)

FMB 6241 ¡ 231 5563 ¡ 342 10.86MFMB 5897 ¡ 134 1872 ¡ 256 68.25

a Values are expressed as mean ¡ SD, n = 3.

Table 4 Pharmacokinetic parameters of NFX following oral administration asfree NFX, FMB and MFMB

Pharmacokinetic parameters Free NFX FMB MFMB

Cmax (mg ml21)a 1.78 ¡ 0.05 1.65 ¡ 0.09 1.48 ¡ 0.09Tmax (h) 1 2 2AUC0-‘ (mg h ml21) 5.3751 7.7615 11.311t1/2 (h) 2.7063 4.5980 5.3489Clearance (ml h21 kg21) 3.7209 2.5768 1.7682Ke (h21) 0.2561 0.1507 0.1296MRT‘ (h) 3.3492 5.9300 7.9471

a (Mean ¡ SEM, n = 5).

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absorbed from the upper GIT and conventional dosage formsrelease low amounts of drug in the preferred absorption regionbefore moving further down in the GIT. Whereas, thegastroretentive system targets the release of drug at the gastriccavity in a sustained manner and the released drug isabsorbed from the upper GIT following the process of passiveabsorption which leads to a higher bioavailability of NFX. TheMFMB showed a prolonged serum level of NFX in comparisonto FMB which implies that MFMB is a superior controlledrelease drug delivery system than FMB. Overall, the results actas a proof that the gastroretentive dual working system canimprove the performance in comparison to gastroretentivedrug delivery systems based on a single approach that is eitherfloating or mucoadhesion and can improve the pharmacother-apy of drugs.

D Conclusion

A gastroretentive dual working system based on floating andmucoadhesion principles of gastroretention was successfullydeveloped and optimized for various parameters. Initially,hollow microballoons bearing NFX as a model drug wereprepared via a solvent evaporation method followed bysubsequent coating of microballoons with mucoadhesivechitosan to improve the gastroretention. The process wassimple, reproducible and can easily be scaled up. Theperformance of developed formulations was assessed forseveral in vitro aspects. Zero-order kinetics was recorded fordrug release from the formulations suggesting that thedeveloped system is suitable for controlled drug release.Chitosan coating over the microballoons enhanced thegastroretention of the microballoons. The in vivo studydemonstrated that the microballoons prolonged the half-lifeof NFX and increased the overall plasma drug concentration.The pharmacokinetics demonstrated significantly improvedbioavailability of NFX in the case of the coated formulation. In

conclusion, the developed prototype mucoadhesive floatinggastroretentive drug delivery system can increase the gastro-retention of the dosage forms in order to improve thebioavailability of NFX with the possibility of a reduction inthe dosing frequency.

Acknowledgements

We are very grateful to Birbal Sahni Institute of Palaeobotanyfor providing the SEM facility. Authors would like to acknowl-edge the financial support received from CSIR network projectSPLenDID.

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