Original article

Chitosanepectin polyelectrolyte complex as a carrier for colontargeted drug delivery

Sonia Pandey Ashish Mishra Pooja Raval Hetal Patel Arti Gupta Dinesh ShahMaliba Pharmacy College Surat Gujarat 394350 India

a r t i c l e i n f o

Article historyReceived 29 August 2013Accepted 16 November 2013Available online 28 December 2013

KeywordsPolyelectrolyte complex (PEC)ChitosanPectinColon targeted drug delivery

a b s t r a c t

Objective The objective of present work was to prepare a polyelectrolyte complex (PEC) between chi-tosan (polycation) amp pectin (polyanion) and to develop enteric coated tablets for colon delivery using thePECMethodology The PECs were prepared using different concentrations of chitosan and pectin Drug loadedenteric coated tablets were prepared by wet granulation method using PEC to sustain the release at colonand coating was done with Eudragit S 100 to prevent the early release of the drug in stomach and in-testine Two independent variable PEC (chitosanpectin) and coating were optimized by 32 fullfactorial design Statistical model were also used to supplement the optimization DSC was performed toconfirm the interaction between the polyions Developed formulations were evaluated for physicalappearance weight variation thickness hardness friability swelling assay in-vitro and ex-vivo drugrelease studies to investigate the PECrsquos ability to deliver the drug to colon Ex-vivo release study using ratcaecal content was also carried out on optimized formulationResults and discussion DSC results confirmed chitosanpectin interaction and subsequent formation ofPEC The optimized formulation containing 11 of PEC and 3 of coating showed highest swelling andrelease in alkaline pH mechanism of which was found to be microbial enzyme dependent degradationestablished by ex-vivo study using rat caecal contentCopyright 2013 InPharm Association Published by Reed Elsevier India Pvt Ltd All rights reserved

1 Introduction

In the development of colon targeted delivery interpolymercomplex (IPC) hydrogels have generated considerable interest asvehicle for drug delivery IPC provides free volume space for theeasy encapsulation of drugs in its three-dimensional networkstructure commonly known as polyelectrolyte complex (PEC) ob-tained by cross-linking of two or more polymers1 The colon pro-vides a site for drug delivery with distinct advantages of a nearneutral pH a much longer transit time relatively low photolyticenzyme activity and greater responsiveness to absorption en-hancers Colon-specific delivery systems would prevent release ofthe drug in the upper-part of GIT but would require a triggeringmechanism to affect an abrupt release on reaching the colon23 Thesuccessful targeted delivery of drugs to the colon via the gastro-intestinal tract (GIT) requires the protection of a drug from degra-dation and release in the stomach and small intestine and then

ensures abrupt or controlled release in the proximal colon4 Variousproperties of IPCs such as porosity bio-adhesiveness elasticityswelling and stimuli-responsive behavior can be controlled by theappropriate choice of the network-forming polymers5 F Bigucciet al (2008) have also earlier prepared chitosanpectin poly-electrolyte complexes for colon-specific delivery of vancomycin butthe present work focuses on economizing the development of drugloaded enteric coated tablets of PEC

Several natural polysaccharides are extensively used to preparethe PEC Chitosan is soluble at low pH of stomach hence there is aneed to make enteric coated formulation that would protect it fromstomachrsquos environment A coating with pH dependent polymerEudragit S100 was done to prevent the early release of drug instomach and intestine As chitosan alone cannot provide sustainedrelease at the site pectin was used for cross-linking to formulatePEC Pectin is highly water soluble absorbed in the upper GIT andcompletely degraded by the colonic bacterial enzymes Accountingfor all properties of PEC and coating polymer tablets were preparedusing various concentrations of PEC The developed formulationshowed pH dependent swelling and release in alkaline pH Ex-vivoconfirmed that polysaccharide degradation by colonic microbialenzyme was the prime source of drug release in colon6

Corresponding author Tel thorn91 9725128861 (mobile) fax thorn91 (0) 2625255882

E-mail address soniapandeyutuacin (S Pandey)

Contents lists available at ScienceDirect

Journal of Young Pharmacists

journal homepage wwwelsevier comlocate jyp

0975-1483$ e see front matter Copyright 2013 InPharm Association Published by Reed Elsevier India Pvt Ltd All rights reservedhttpdxdoiorg101016jjyp201311002

Journal of Young Pharmacists 5 (2013) 160e166

2 Materials and methods

21 Materials

Theophylline was a gift from Sidmak Pharma Vapi India Lowmolecularweight (LMW) chitosan (molecularweightfrac14 50e190 kDa)was obtained from Balaji Drugs Ltd Mumbai India Pectin sodiumhydroxide glacial acetic acid sodium acetate sodium starch glyco-late (SSG) potassium dihydrogen phosphate magnesium stearatewere obtained from SD Fine Chemicals Ltd Erythrosine was boughtfrom Yarrow Chemicals Ltd India Talc was obtained from Chem PortPte Ltd India All other chemicals used were of analytical grade

22 Drug e excipients compatibility study

221 Fourier transform infrared spectroscopy (FT-IR)Compatibility of theophylline with the individual excipients was

established by FT-IR Any change in the chemical composition aftercombining with excipients was investigated with IR spectral anal-ysis The IR spectra of drug and drug thorn excipients were recorded inFT-IR (Bruker Optics Alpha) in the range of 4000e500 cm1

23 Preparation of chitosanpectin IPC

Chitosan solution (1 wv) was prepared in acetic acid Pectinsolution (5 wv) was prepared in distilled water Both the solu-tions were sonicated separately till their clear solutions were ob-tained After that both solutions were mixed agitated for 30 minand then kept for drying for 5 h to yield a dry powder

24 Characterization of IPC

241 By DSCChitosan pectin and PEC were subjected to DSC study using

Shimadzu DSC 60 (Kyoto Japan) 10 mg sample was heated inaluminum pan at a rate of 20 Cmin in the temperature range of50e300 C under the nitrogen flow of 40 mlmin

25 Tablet preparation

Drug and all excipients except PEC were passed through the 60 sieve and then PEC was mixed Dry mass was granulated usingisopropyl alcohol The granules so obtained were dried at 50 C for1 h in the oven Dried granules were passed through 22 sieve andfines were separated using 44 sieve SSGwas passed through 60 sieve and mixed with dried granules These granules were lubri-cated with magnesium stearate and talc The lubricated granuleswere compressed into tablets using tablet compression machine(Rimek Mini Press-I India) Weight variation hardness friabilityand disintegration test were performed for the core tabletsFormulation composition of the tablet is given in Table 1

26 Preparation of coating solution

Coating solution was made by dissolving Eudragit S100 inisopropyl alcohol A bath sonicator was used to homogenize thecoating solution After sonication PEG 400 (125vv) as plasticizer

was added to the solution The coating solution so prepared wasapplied on tablets by intermittently spraying it through a pilot typespray gun fitted with 1 mm spray nozzle in pan coater (Hardik

Manufacturers India) The spray dispersion was maintained at arate of 4e8 mlmin and bed temperature of 35e40 C at 35 rpmThe 32 full factorial designwas employed inwhich two factors wereevaluated each at 3 levels and experimental trials were performedat all 9 possible combinations The amount of PEC and coatingwere selected as independent variables while drug release at 4 h(Q4) and at 12 h (Q12) were selected as dependent variables (Table2a and b)

As shown in Eq (1) a statistical model incorporating interactiveand polynomial terms was used to evaluate the responses

Y frac14 b0 thorn b1X1 thorn b2X2 thorn b3X1X2 thorn b4X12 thorn b5X2

2 thorn b6X12X2 thorn b7X1X2

2 thorn b8 X12X2

2 (1)

where Y is dependent variable b0 is arithmetic mean response b1is coefficient of factor X1 The main effects X1 and X2 represent theaverage result of changing one factor at a time from its low to highvalue The interaction terms X1X2 X2

1X2 X1X22 and X2

1X22 show

how the dependent variable changes when two or more factors aresimultaneously changed Statistical analysis was done usingMicrosoft Excel 2007 Contour plots and response surface plotswere drawn using Design Expert software7e9

27 Evaluation of core tablets

Core tablets were evaluated for appearance diameter thicknessweight variation hardness friability hardness disintegration drugcontent and content uniformity test Weight variation test anddisintegration test were performed as per USP1011 Hardnessthickness and friability were determined by Monsanto HardnessTester Digital Vernier Calipers and Roche Friabilator respectively

271 Swelling studyThree tablets from each batch were weighted accurately and

immersed in dissolution medium (100 ml of 01 N HCl) for 2 h Fornext 1 h tablets were kept in acetate buffer (pH 46) after that fornext 4 h tablets were placed in phosphate buffer (pH 68) and thenonwards tablets were placed in phosphate buffer (pH 74) for next5 h Tablets were taken out carefully after each time interval up to12 h blotted with filter paper to remove the fluid present on thesurface and weighed accurately Percentage swelling (swelling in-dex) was calculated using the following formula12

272 Drug release study2721 In-vitro drug release study of Eudragit S 100 coated tabletsIn-vitro dissolution studies of coated tablets were carried out using

Table 1Tablet composition

Ingredient Quantity

Theophylline 100 mgSodium starch glycolate 4 wwChitosanpectin IPC (15) (ww) 1 2 3Magnesium stearate 002 gTalc 001 gLactose monohydrate qs 400 mg

Swelling index frac14 Wet weight of tablet Dry weight of tabletDry weight of tablet

100 (2)

S Pandey et al Journal of Young Pharmacists 5 (2013) 160e166 161

dissolution test apparatus USP I paddle type The dissolutionmediumconsisted of 900 ml of pH 12 for first 2 h followed by pH 46 and 68phosphate buffers for 1 and 3 h respectively and finally pH 74 phos-phate buffer for the remaining time period up to 12 h The tempera-ture of the mediumwas maintained at 37 05 C and the speed ofrotation of the basket was 50 rpm Aliquots of 10ml were withdrawnafter every hour and replaced with the fresh dissolution mediumequilibrated at the same temperature The absorbance of the sampleswas determined at wavelength according to the dissolution mediumpH12 (lmaxfrac14 263 nm) 46 (lmaxfrac14 271 nm) 68 (lmaxfrac14 272 nm) and74 (lmax frac14 267 nm) using UVeVisible spectrophotometer (UV-1800Shimadzu) against respective buffer solutions as a blank

28 Release drug data modeling

281 KorsmeyerePeppas model13

MtMN

frac14 ktn (3)

where k is the rate constant and n is release exponent that char-acterizes the mechanism of drug release For cylindrical matrixtablets if n frac14 045 it indicates drug release mechanism by Fickiandiffusion and if 045 lt n lt 089 it is non-Fickian or anomalousdiffusion

29 Ex-vivo release study

The drug release studies were carried out using USP dissolutionrate test apparatus (apparatus 1 100 rpm 37 05 C) Each for-mulations after completing the dissolution study in 01 N HCl (2 h)acetate buffer pH 46 (1 h) and phosphate buffer pH 68 (3 h) wasplaced in the basket of the apparatus and immersed in 4 rat cecalcontent medium (phosphate buffer pH 74) At fixed time intervals10 ml of sample was withdrawn replaced with 10 ml of freshphosphate buffer pH 74 and the experiment was continued foranother 6 h Sample was filtered and analyzed for theophylline byUV Spectrophotometer at 263 nm 271 nm 272 nm 267 nm for01 N HCl acetate buffer pH 46 phosphate buffer pH 68 andphosphate buffer pH 74 respectively 14

3 Results and discussion

31 By FT-IR

The FT-IR spectra of theophylline excipient mixture andtheophylline and excipient mixture have been shown in Fig 1 Thespectra exhibited no change in the absorption peaks of theophyllinevis-agrave-vis the theophylline thorn excipient mix so it can be concludedthat there is no interaction between the components

32 Characterization of prepared IPC

321 By DSCThe chitosan showed the first melting point peak at 567 C and

second at 312 C Pectin showed broad peak at 238 C DSC curve ofPEC mixture (IPC) of chitosan and pectin showed peak at 215 CThe results of DSC are in compliance with observations of Ghaffariet al15 who had reported interaction (chitosan and pectin) tem-perature in the range of 210e220 C The shift to lower temperaturein melting point peak of the complex indicates that the probableformation of ionic bonds between chitosan and pectin is correlatedto the loss of organization (Fig 2)16

Table 2b32 Full factorial design matrix with interaction terms

Batch X1 X2 X12 X11 X22

F1 1 1 thorn1 thorn1 thorn1F2 0 1 0 0 thorn1F3 thorn1 1 1 thorn1 thorn1F4 1 0 0 thorn1 0F5 0 0 0 0 0F6 thorn1 0 0 thorn1 0F7 1 thorn1 1 thorn1 thorn1F8 0 thorn1 0 0 thorn1F9 thorn1 thorn1 thorn1 thorn1 thorn1

Table 2aLayout of factorial design

Translation of coded values in actual units

Independent variables Levels used actual (coded)

Low (1) Medium (0) High (thorn1)

X1 frac14 amount of PEC 1 2 3X2 frac14 of coating 3 5 7Dependent variables1 Percentage cumulative release at 4 h (Q4)2 Percentage cumulative release at 12 h (Q12)

Fig 1 FT-IR spectra of theophylline and excipient mixture

S Pandey et al Journal of Young Pharmacists 5 (2013) 160e166162

322 Evaluation of physical properties of tabletsTable 3 lists the physical properties (thickness diameter weight

hardness friability) and assay of tablet They produced with smallweight variations (less than 6) uniform thickness and hardnessacceptable friability (less than 1) and drug content The averageweight of tablet formulation was within the range of 3876 0028to 4107 0091 mg The hardness of the tablets ranged between69 014 to74 014 kgcm2 In case of content uniformity testdrug contentwas found to bewithin 955 003 to 9823 0023of labeled amount

323 Swelling studiesThe enteric coated tablets showed pH sensitive swelling

behavior The degree of swelling is mainly affected by pH of themedium type of anionic polymer and amount of IPC (chitosan andpectin) As pH of the swelling medium changes (12 46 68 and 74)the degree of interaction between chitosan and pectin is modifiedand swelling occurs because of the dissociation of the complex Inacidic medium pectin is neutralized and free positive charge(NH3thorn) appears inside the gel while in basic medium chitosan isneutralized and free negative charge (COO) appears inside the gelThe mutual repulsion between positive or negative charges and theentry of water together with counterions to neutralize thesecharges cause swelling All the tablets showed low swelling in pH12 (01 N HCl) pH 46 (acetate buffer) and pH 68 (phosphatebuffer) In pH 74 (phosphate buffer) the degree of swelling wasincreased due to deprotonation of chitosan It was observed that asthe concentration of IPC increased the swelling index decreasedBatch F4 (2 IPC 5 coating) showed maximum swelling index of7951161 Thus IPC tablet is hydrophilic and swells considerablyin phosphate buffer solutions This process shows a pH-dependentpattern There also exists the possibility of intramolecular hydrogenbonding between the OH or COOCH3 groups within the network of

the IPC However at pH 74 the eNH2 group of chitosan will bepartially unionized Therefore at this pH the IPC network will beloose as a result of suboptimal NH3

thorn- eOOC ionic interaction Inother words stronger ionic interaction may result in a tightening ofthe IPC network leading to a reduced swelling capacity at pH 68 ascompared with pH 74

324 Evaluation of enteric coated tabletsTablets weight coating and disintegration time of coated sec-

ond pulse tablets are reported in Table 4Weight variation of entericcoated tablets for all batches (F1 to F9) was below 3 The weightgain calculated from the average tablet weight for Coating wasfound to be in the range of 3e7 In the disintegration test all thetablets remained intact in 01NHCl for a 1 h and showedno cracks orfracture Same tablets disintegrated within 45 min in pH 74 indi-cating that the disintegration time in pH 74 increasedwith increasein weight Eudragit S 100was used in the preparation to target thecolon Isopropyl alcohol was used as a solvent for the coatingdispersion due to its high flash point (15) lowboiling point (823)and low heat of evaporation (667 Jg) Eudragit is a brittle polymerhence it was needed to lower the glass transition temperature ofpolymer and promote formation of a good elastic film Polymersoftening tendencies stimulate sticking at higher temperature sothe bed temperature was maintained at 35e40 C using an infraredlamp To getfine spraydroplets the spraynozzle of 1mmwasused atan atomization air pressure of 4 kgcm2 By controlling the rotatingpan at speed of 20 rpm and the rate of spray dispersion applicationthe sticking tendency of the tablets was overcome

325 Drug release study3251 In-vitro drug release of factorial batches All the formula-tions were evaluated for in-vitro drug release The cumulative theophylline release from all 9 formulations as the function of thetime is shown in Fig 3

Cumulative drug release profile of factorial batches (F1 to F9)showed sigmoidal drug release pattern (Fig 3) The in-vitro releaseof drug from formulations was mainly affected by concentration of

Fig 2 DSC graph of pectin chitosan IPC

Table 3Evaluation parameters of tablet

Batch Average weight(mg) (n frac14 20)

Hardness (kgcm2)(n frac14 3)

Friability ()(n frac14 6)

Thickness (mm)(n frac14 6)

Diameter (mm)(n frac14 6)

Assay () (n frac14 3) Disintegrationtime(min) (n frac14 6)

F1 3926 0018 708 014 037 021 426 0023 1027 0009 993 032 35 0124F2 4107 0091 717 029 033 034 426 0040 1029 0015 991 073 38 0125F3 3993 0012 692 014 043 023 425 0016 1029 0010 9954 036 37 0124F4 3876 0028 717 014 033 025 425 0024 1030 0010 9952 037 36 0123F5 4201 01 7 00 058 031 424 0023 1029 0005 9957 017 39 0127F6 4161 001 742 014 050 026 425 0026 1030 0007 9959 021 40 0127F7 4009 0085 692 014 029 036 427 0012 1029 0014 9872 011 41 0128F8 4034 0018 708 029 041 038 425 0040 1029 0007 9964 018 38 0126F9 3868 0056 750 00 070 035 424 0030 1029 0009 9868 027 37 0127

Table 4Evaluation of enteric coated tablets

Batch code Weight of tableta

(mg)(n frac14 20) Coating Disintegration time in

pH 74 phosphate buffer

F1 41225 162 3 25 min 58 sF2 42032 125 5 32 min 42 sF3 42886 154 7 38 min 21 sF4 41214 024 3 28 min 34 sF5 42028 053 5 35 min 28 sF6 42858 018 7 40 min 14 sF7 41221 023 3 30 min 84 sF8 42038 075 5 38 min 54 sF9 42867 090 7 44 min 25 s

a Value expressed as mean SD

S Pandey et al Journal of Young Pharmacists 5 (2013) 160e166 163

dissolution test apparatus USP I paddle type The dissolutionmediumconsisted of 900 ml of pH 12 for first 2 h followed by pH 46 and 68phosphate buffers for 1 and 3 h respectively and finally pH 74 phos-phate buffer for the remaining time period up to 12 h The tempera-ture of the mediumwas maintained at 37 05 C and the speed ofrotation of the basket was 50 rpm Aliquots of 10ml were withdrawnafter every hour and replaced with the fresh dissolution mediumequilibrated at the same temperature The absorbance of the sampleswas determined at wavelength according to the dissolution mediumpH12 (lmaxfrac14 263 nm) 46 (lmaxfrac14 271 nm) 68 (lmaxfrac14 272 nm) and74 (lmax frac14 267 nm) using UVeVisible spectrophotometer (UV-1800Shimadzu) against respective buffer solutions as a blank

28 Release drug data modeling

281 KorsmeyerePeppas model13

MtMN

frac14 ktn (3)

where k is the rate constant and n is release exponent that char-acterizes the mechanism of drug release For cylindrical matrixtablets if n frac14 045 it indicates drug release mechanism by Fickiandiffusion and if 045 lt n lt 089 it is non-Fickian or anomalousdiffusion

29 Ex-vivo release study

The drug release studies were carried out using USP dissolutionrate test apparatus (apparatus 1 100 rpm 37 05 C) Each for-mulations after completing the dissolution study in 01 N HCl (2 h)acetate buffer pH 46 (1 h) and phosphate buffer pH 68 (3 h) wasplaced in the basket of the apparatus and immersed in 4 rat cecalcontent medium (phosphate buffer pH 74) At fixed time intervals10 ml of sample was withdrawn replaced with 10 ml of freshphosphate buffer pH 74 and the experiment was continued foranother 6 h Sample was filtered and analyzed for theophylline byUV Spectrophotometer at 263 nm 271 nm 272 nm 267 nm for01 N HCl acetate buffer pH 46 phosphate buffer pH 68 andphosphate buffer pH 74 respectively 14

3 Results and discussion

31 By FT-IR

The FT-IR spectra of theophylline excipient mixture andtheophylline and excipient mixture have been shown in Fig 1 Thespectra exhibited no change in the absorption peaks of theophyllinevis-agrave-vis the theophylline thorn excipient mix so it can be concludedthat there is no interaction between the components

32 Characterization of prepared IPC

321 By DSCThe chitosan showed the first melting point peak at 567 C and

second at 312 C Pectin showed broad peak at 238 C DSC curve ofPEC mixture (IPC) of chitosan and pectin showed peak at 215 CThe results of DSC are in compliance with observations of Ghaffariet al15 who had reported interaction (chitosan and pectin) tem-perature in the range of 210e220 C The shift to lower temperaturein melting point peak of the complex indicates that the probableformation of ionic bonds between chitosan and pectin is correlatedto the loss of organization (Fig 2)16

Table 2b32 Full factorial design matrix with interaction terms

Batch X1 X2 X12 X11 X22

F1 1 1 thorn1 thorn1 thorn1F2 0 1 0 0 thorn1F3 thorn1 1 1 thorn1 thorn1F4 1 0 0 thorn1 0F5 0 0 0 0 0F6 thorn1 0 0 thorn1 0F7 1 thorn1 1 thorn1 thorn1F8 0 thorn1 0 0 thorn1F9 thorn1 thorn1 thorn1 thorn1 thorn1

Table 2aLayout of factorial design

Translation of coded values in actual units

Independent variables Levels used actual (coded)

Low (1) Medium (0) High (thorn1)

X1 frac14 amount of PEC 1 2 3X2 frac14 of coating 3 5 7Dependent variables1 Percentage cumulative release at 4 h (Q4)2 Percentage cumulative release at 12 h (Q12)

Fig 1 FT-IR spectra of theophylline and excipient mixture

S Pandey et al Journal of Young Pharmacists 5 (2013) 160e166162

322 Evaluation of physical properties of tabletsTable 3 lists the physical properties (thickness diameter weight

hardness friability) and assay of tablet They produced with smallweight variations (less than 6) uniform thickness and hardnessacceptable friability (less than 1) and drug content The averageweight of tablet formulation was within the range of 3876 0028to 4107 0091 mg The hardness of the tablets ranged between69 014 to74 014 kgcm2 In case of content uniformity testdrug contentwas found to bewithin 955 003 to 9823 0023of labeled amount

323 Swelling studiesThe enteric coated tablets showed pH sensitive swelling

behavior The degree of swelling is mainly affected by pH of themedium type of anionic polymer and amount of IPC (chitosan andpectin) As pH of the swelling medium changes (12 46 68 and 74)the degree of interaction between chitosan and pectin is modifiedand swelling occurs because of the dissociation of the complex Inacidic medium pectin is neutralized and free positive charge(NH3thorn) appears inside the gel while in basic medium chitosan isneutralized and free negative charge (COO) appears inside the gelThe mutual repulsion between positive or negative charges and theentry of water together with counterions to neutralize thesecharges cause swelling All the tablets showed low swelling in pH12 (01 N HCl) pH 46 (acetate buffer) and pH 68 (phosphatebuffer) In pH 74 (phosphate buffer) the degree of swelling wasincreased due to deprotonation of chitosan It was observed that asthe concentration of IPC increased the swelling index decreasedBatch F4 (2 IPC 5 coating) showed maximum swelling index of7951161 Thus IPC tablet is hydrophilic and swells considerablyin phosphate buffer solutions This process shows a pH-dependentpattern There also exists the possibility of intramolecular hydrogenbonding between the OH or COOCH3 groups within the network of

the IPC However at pH 74 the eNH2 group of chitosan will bepartially unionized Therefore at this pH the IPC network will beloose as a result of suboptimal NH3

thorn- eOOC ionic interaction Inother words stronger ionic interaction may result in a tightening ofthe IPC network leading to a reduced swelling capacity at pH 68 ascompared with pH 74

324 Evaluation of enteric coated tabletsTablets weight coating and disintegration time of coated sec-

ond pulse tablets are reported in Table 4Weight variation of entericcoated tablets for all batches (F1 to F9) was below 3 The weightgain calculated from the average tablet weight for Coating wasfound to be in the range of 3e7 In the disintegration test all thetablets remained intact in 01NHCl for a 1 h and showedno cracks orfracture Same tablets disintegrated within 45 min in pH 74 indi-cating that the disintegration time in pH 74 increasedwith increasein weight Eudragit S 100was used in the preparation to target thecolon Isopropyl alcohol was used as a solvent for the coatingdispersion due to its high flash point (15) lowboiling point (823)and low heat of evaporation (667 Jg) Eudragit is a brittle polymerhence it was needed to lower the glass transition temperature ofpolymer and promote formation of a good elastic film Polymersoftening tendencies stimulate sticking at higher temperature sothe bed temperature was maintained at 35e40 C using an infraredlamp To getfine spraydroplets the spraynozzle of 1mmwasused atan atomization air pressure of 4 kgcm2 By controlling the rotatingpan at speed of 20 rpm and the rate of spray dispersion applicationthe sticking tendency of the tablets was overcome

325 Drug release study3251 In-vitro drug release of factorial batches All the formula-tions were evaluated for in-vitro drug release The cumulative theophylline release from all 9 formulations as the function of thetime is shown in Fig 3

Cumulative drug release profile of factorial batches (F1 to F9)showed sigmoidal drug release pattern (Fig 3) The in-vitro releaseof drug from formulations was mainly affected by concentration of

Fig 2 DSC graph of pectin chitosan IPC

Table 3Evaluation parameters of tablet

Batch Average weight(mg) (n frac14 20)

Hardness (kgcm2)(n frac14 3)

Friability ()(n frac14 6)

Thickness (mm)(n frac14 6)

Diameter (mm)(n frac14 6)

Assay () (n frac14 3) Disintegrationtime(min) (n frac14 6)

F1 3926 0018 708 014 037 021 426 0023 1027 0009 993 032 35 0124F2 4107 0091 717 029 033 034 426 0040 1029 0015 991 073 38 0125F3 3993 0012 692 014 043 023 425 0016 1029 0010 9954 036 37 0124F4 3876 0028 717 014 033 025 425 0024 1030 0010 9952 037 36 0123F5 4201 01 7 00 058 031 424 0023 1029 0005 9957 017 39 0127F6 4161 001 742 014 050 026 425 0026 1030 0007 9959 021 40 0127F7 4009 0085 692 014 029 036 427 0012 1029 0014 9872 011 41 0128F8 4034 0018 708 029 041 038 425 0040 1029 0007 9964 018 38 0126F9 3868 0056 750 00 070 035 424 0030 1029 0009 9868 027 37 0127

Table 4Evaluation of enteric coated tablets

Batch code Weight of tableta

(mg)(n frac14 20) Coating Disintegration time in

pH 74 phosphate buffer

F1 41225 162 3 25 min 58 sF2 42032 125 5 32 min 42 sF3 42886 154 7 38 min 21 sF4 41214 024 3 28 min 34 sF5 42028 053 5 35 min 28 sF6 42858 018 7 40 min 14 sF7 41221 023 3 30 min 84 sF8 42038 075 5 38 min 54 sF9 42867 090 7 44 min 25 s

a Value expressed as mean SD

S Pandey et al Journal of Young Pharmacists 5 (2013) 160e166 163

322 Evaluation of physical properties of tabletsTable 3 lists the physical properties (thickness diameter weight

hardness friability) and assay of tablet They produced with smallweight variations (less than 6) uniform thickness and hardnessacceptable friability (less than 1) and drug content The averageweight of tablet formulation was within the range of 3876 0028to 4107 0091 mg The hardness of the tablets ranged between69 014 to74 014 kgcm2 In case of content uniformity testdrug contentwas found to bewithin 955 003 to 9823 0023of labeled amount

323 Swelling studiesThe enteric coated tablets showed pH sensitive swelling

behavior The degree of swelling is mainly affected by pH of themedium type of anionic polymer and amount of IPC (chitosan andpectin) As pH of the swelling medium changes (12 46 68 and 74)the degree of interaction between chitosan and pectin is modifiedand swelling occurs because of the dissociation of the complex Inacidic medium pectin is neutralized and free positive charge(NH3thorn) appears inside the gel while in basic medium chitosan isneutralized and free negative charge (COO) appears inside the gelThe mutual repulsion between positive or negative charges and theentry of water together with counterions to neutralize thesecharges cause swelling All the tablets showed low swelling in pH12 (01 N HCl) pH 46 (acetate buffer) and pH 68 (phosphatebuffer) In pH 74 (phosphate buffer) the degree of swelling wasincreased due to deprotonation of chitosan It was observed that asthe concentration of IPC increased the swelling index decreasedBatch F4 (2 IPC 5 coating) showed maximum swelling index of7951161 Thus IPC tablet is hydrophilic and swells considerablyin phosphate buffer solutions This process shows a pH-dependentpattern There also exists the possibility of intramolecular hydrogenbonding between the OH or COOCH3 groups within the network of

the IPC However at pH 74 the eNH2 group of chitosan will bepartially unionized Therefore at this pH the IPC network will beloose as a result of suboptimal NH3

thorn- eOOC ionic interaction Inother words stronger ionic interaction may result in a tightening ofthe IPC network leading to a reduced swelling capacity at pH 68 ascompared with pH 74

324 Evaluation of enteric coated tabletsTablets weight coating and disintegration time of coated sec-

ond pulse tablets are reported in Table 4Weight variation of entericcoated tablets for all batches (F1 to F9) was below 3 The weightgain calculated from the average tablet weight for Coating wasfound to be in the range of 3e7 In the disintegration test all thetablets remained intact in 01NHCl for a 1 h and showedno cracks orfracture Same tablets disintegrated within 45 min in pH 74 indi-cating that the disintegration time in pH 74 increasedwith increasein weight Eudragit S 100was used in the preparation to target thecolon Isopropyl alcohol was used as a solvent for the coatingdispersion due to its high flash point (15) lowboiling point (823)and low heat of evaporation (667 Jg) Eudragit is a brittle polymerhence it was needed to lower the glass transition temperature ofpolymer and promote formation of a good elastic film Polymersoftening tendencies stimulate sticking at higher temperature sothe bed temperature was maintained at 35e40 C using an infraredlamp To getfine spraydroplets the spraynozzle of 1mmwasused atan atomization air pressure of 4 kgcm2 By controlling the rotatingpan at speed of 20 rpm and the rate of spray dispersion applicationthe sticking tendency of the tablets was overcome

325 Drug release study3251 In-vitro drug release of factorial batches All the formula-tions were evaluated for in-vitro drug release The cumulative theophylline release from all 9 formulations as the function of thetime is shown in Fig 3

Cumulative drug release profile of factorial batches (F1 to F9)showed sigmoidal drug release pattern (Fig 3) The in-vitro releaseof drug from formulations was mainly affected by concentration of

Fig 2 DSC graph of pectin chitosan IPC

Table 3Evaluation parameters of tablet

Batch Average weight(mg) (n frac14 20)

Hardness (kgcm2)(n frac14 3)

Friability ()(n frac14 6)

Thickness (mm)(n frac14 6)

Diameter (mm)(n frac14 6)

Assay () (n frac14 3) Disintegrationtime(min) (n frac14 6)

F1 3926 0018 708 014 037 021 426 0023 1027 0009 993 032 35 0124F2 4107 0091 717 029 033 034 426 0040 1029 0015 991 073 38 0125F3 3993 0012 692 014 043 023 425 0016 1029 0010 9954 036 37 0124F4 3876 0028 717 014 033 025 425 0024 1030 0010 9952 037 36 0123F5 4201 01 7 00 058 031 424 0023 1029 0005 9957 017 39 0127F6 4161 001 742 014 050 026 425 0026 1030 0007 9959 021 40 0127F7 4009 0085 692 014 029 036 427 0012 1029 0014 9872 011 41 0128F8 4034 0018 708 029 041 038 425 0040 1029 0007 9964 018 38 0126F9 3868 0056 750 00 070 035 424 0030 1029 0009 9868 027 37 0127

Table 4Evaluation of enteric coated tablets

Batch code Weight of tableta

(mg)(n frac14 20) Coating Disintegration time in

pH 74 phosphate buffer

F1 41225 162 3 25 min 58 sF2 42032 125 5 32 min 42 sF3 42886 154 7 38 min 21 sF4 41214 024 3 28 min 34 sF5 42028 053 5 35 min 28 sF6 42858 018 7 40 min 14 sF7 41221 023 3 30 min 84 sF8 42038 075 5 38 min 54 sF9 42867 090 7 44 min 25 s

a Value expressed as mean SD

S Pandey et al Journal of Young Pharmacists 5 (2013) 160e166 163

chitosan types of anionic polymers (pectin) used coating andswelling behavior of the polymer Thus in turn it mainly dependsupon the amount of IPC used The in-vitro release study was per-formed in HCl buffer (pH 12) for initial first 2 h Then the mediumwas replaced by acetate buffer (pH 46) for 1 h followed by phos-phate buffer (pH 68) for 3 h Study was continued for next 6 h inphosphate buffer (pH 74) Eudragit S100 is soluble at pH near 70hence it dissolves at pH 74 and leads to formation of porous surfaceat the coating layer which allows medium to diffuse into the coretablet to ruptures outer coat Thus the level of coating plays a veryimportant role for in optimizing the formulation At pH 12 and pH46 none of the formulations showed any release of drug due toenteric coating while some batches started releasing the drug atpH 68 The release of drugwas amaximum of 9090 from batch F4(2 IPC 5 coating) near to pH 7 at colon site The ionic interactionbetween chitosan and negatively charged pectin was greatlyreduced at colonic pH forming a loose network This increasedporous surface allowed entry of large amount of dissolution medialeading to complete release of drug Hence it can be assumed thatat pH 74 of phosphate buffer rapid dissociation of IPC membraneoccurs which leads to drug release

326 Drug release data modelingThe mechanism of drug release from enteric coated tablets

containing PEC is complicated and not completely understoodSome systems may be classified as either diffusion or erosioncontrolled while the most systems exhibit a combination of thesemechanisms17 The drug release data of all the 9 batches weremathematically analyzed using KorsmeyerePeppas semi-empirical model equation The linearity of the model was evalu-ated by calculating the linear correlation coefficient (r2) while therelease mechanism was determined by evaluating the releaseexponent (n)

Coefficients of correlation (r2) were used to evaluate the accu-racy of the fit The r2 and n values are given in Table 5 The r2 valuesranged between 09460 and 09759 for all the formulations The n

value were in the range of 0555e0686 (ie 05 lt n lt 1) indicatingthat the release mechanism of theophylline from these matrices isan anomalous (non-Fickian) transport which suggests that bothdiffusion of the drug from the hydrated matrix and its own erosionmodulate drug release1819

327 Data analysis3271 Data analysis for drug release at 4 h (Q4) There was notmuch difference between actual and predicted values for all 9batches r2 Value in plot of predicted vs observed responses was09899 which indicated excellent fit The response (Y1) obtained atthree levels of the two independent variables (X1 and X2) weresubjected to multiple regression to yield a polynomial Eq (4)Equation clearly reflects the wide range of values for coefficients (b)

Q4 frac14 011 thorn 047X1 017X2 022X1X2 thorn 080X12 030X22 (4)

In the present study coefficients b1 and b2 possessed positiveand negative sign indicating synergistic and antagonistic effect ofvariables X1 and X2 on response Y1 (Q4) Among two independentvariables X1 (amount of PEC) has prominent effect (b1 frac14 047 andp frac14 00022) on Q4 whereas to some extent X2 ( coating) also af-fects the results (b2 frac14 017 and p frac14 01299) in inverse mannerSignificance (F value) is less than 005 The high values of the co-efficient of determination indicate a good fit ie good agreementbetween the dependent and independent variables The co-efficients b1 was found to be significant at plt 005 but b2 was foundto be insignificant at p gt 005

3272 Data analysis for drug release at 12 h (Q12) The r2 value forQ12 in plot of predicted vs observed responses was 09593 whichindicated excellent fit The Y2 (Q12) values observed for differentbatches showed wide variation ie values ranged from a minimumof 4474 234 to a maximum of 9090 225 There was notmuch difference between actual and predicted values Eq (5) refersto polynomial equation (full model) for response Y2

Q12 frac14 0105 thorn 0474X1 0168X2 0217X1X2 thorn 0797X12 0297X22 (5)

Coefficient b1 and b2 possessed positive and negative signrespectively which indicated positive effect of X1 and antagonisticeffect of X2 variables on response Y2 (Q12) Independent variablesX1 (amount of cross-linking) (b1 frac14 0474 and p frac14 00039) affectsmore prominently the Q12 then X2 ( coating) (b2 frac14 0168 andpfrac14 04685) which has inverse effect on Q12 Significance (F value) isless than 005 The coefficient b1 was found to be significant atp lt 005 and b2 was found to be insignificant at p gt 005 Table 6

Two-dimensional contour plots and three-dimensionalresponse surface plots are presented in Fig 4aed which are use-ful tool to study interaction effects of the factors on responsesFig 4a and c exhibited non-linear pattern but with an increase inamount of PEC and of coating it also showed that amount of PEChas a comparatively greater influence on response variable Q4 then of coating In contrast to the result of drug release at 4 h contourplot of drug release in 12 h varies in non-linear manner with anincrease in amount of PEC and of coating (Fig 4b and d) Howeverthe effect of X1 (amount of PEC) seems to be more pronounced ascompared with that of coating The results was also confirmedthrough 3D response surface graph

328 Selection of optimized formulationFrom the polynomial equation and the contour plots the opti-

mized batch T1 was found The optimum formulation was selectedbased on the criteria of attaining the constraints of variables

Fig 3 In-vitro dissolution of all formulations

Table 5Results of kinetic model fitting for factorial batches (F1eF9)

Batch KorsmeyerePeppas

r2 n

F1 09595 0686F2 09460 0692F3 09516 0632F4 09749 0614F5 09759 0650F6 09636 0637F7 09180 0620F8 09518 0612F9 09514 0555

S Pandey et al Journal of Young Pharmacists 5 (2013) 160e166164

response Upon lsquotrading ofrsquo various response variables andcomprehensive evaluation of feasibility search and exhaustive gridsearch the formulation compositionwith amount of PEC (11) andcoating (3) was found to fulfill the requisite of an optimum for-mula In-vitro release data of optimized formulation are showed92 theophylline release upon completion of 12 h but only 0096release in initial first 4 h Composition of the optimized batch T1was also very economical as compared to the next best batch F4obtained from factorization

329 Validation of response surface methodologyTwo check points were selected for validation of response sur-

face methodology from contour and response surface plots For allof the 2 checkpoint formulations the results of the dependentvariables (Q4 and Q12) were found to be within limits

33 Ex-vivo release data

The drug release data in presence of rat caecal contents areshown in Fig 5 Rat caecal microflora was used because of theinherent similarity with human intestinal microflora The main aimof the drug delivery system targeted to the colon is not only toprotect the drug from being released in the physiological

environment of stomach and small intestine but also to release thedrug in the colon after enzymatic degradation of polysaccharideHence the in-vitro drug release studies were carried out in phos-phate buffer (pH 68) and phosphate buffer (pH 74) containing 4wv of rat caecal content Optimized batch T1 shows 9858 drugrelease in presence of rat caecal content There was increase in drugrelease in the dissolution medium containing rat cecal content (4)as compared to control group (without rat caecal content)

4 Conclusion

Chitosanepectin polyelectrolyte complex was prepared bysimple interaction of positively charged chitosan with negativelycharged pectin The interaction was confirmed by DSC The opti-mized batch consisting of PEC (11) and coating (3) showed9216 drug release within 12 h The ex-vivo drug release was foundto be 9858 192 in presence of rat caecal content

Fig 4 (a) Contour plot Q4 (b) contour plot Q12 (c) response surface of Q4 (d) response surface of Q12

Fig 5 Drug release profile of optimized formulation with and without rat cecal con-tent (No drug release in presence of rat caecal content was observed in 01 N HCl andphosphate buffer 46 after 3 h)

Table 6Summary of results of multiple regression analysis of Q4 and Q12

Dependent variable Q4 frac14 Y1 Q12 frac14 Y2

P value Coefficient P value Coefficient

Intercept 00030 011 00390 6757X1 00022 047 00039 1443X2 01399 017 04685 263X12 01299 022 04081 368

S Pandey et al Journal of Young Pharmacists 5 (2013) 160e166 165

Conflicts of interest

All authors have none to declare

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