Impact of degree of substitution of acetylated Ofada rice...

Impact of degree of substitution of acetylated Ofada rice starchpolymer on the release properties of nimesulide microspheres .

Adenike Okunlola*, Temitope Owojori

Department of Pharmaceutics and Industrial Pharmacy, University of Ibadan, Ibadan, Nigeria

Received: January 22, 2016; Accepted: February 13, 2016 Original Article

ABSTRACT

Nimesulide microspheres were prepared by the quasi-emulsion solvent diffusion method, using acetylatedstarches of the indigenous Ofada rice (Oryza glaberrima Steud) with degrees of substitution (DS) 1.42 and 2.62.A full 23 factorial experimental design was performed using DS (X1), drug:polymer ratio (X2) and polymerconcentration (X3) as independent factors; size, entrapment, swelling and time taken for 80% drug release (t80)were the dependent variables. Contour plots were generated and data from the in vitro release studies werefitted to various kinetic models. Nimesulide microspheres were near-spherical, sizes varying from50.91±16.22 to 74.24±24.73 µm for microspheres containing starch DS 1.42 and from 21.05±4.25 to46.10±3.85 µm for starch DS 2.62. Drug entrapment was 56.75±0.45 to 98.28±2.30%. DS had the greatesteffect on the size, swelling and dissolution time (p = 0.01) which was confirmed by the contour plots. Theinteraction between factors DS and drug:polymer ratio (X1X2) had the greatest effect on the microsphereproperties (p = 0.04). Drug release was fitted into the First Order, Higuchi and Korsmeyer models. Acetylatedstarch of Ofada rice DS 2.62 was found more suitable for the formulation of microspheres because ofreduced size and swelling, higher entrapment and prolonged drug release.

KEY WORDS: Acetylation, degree of substitution, factorial design, Nimesulide, Ofada rice starch

INTRODUCTION

The coating polymer materials used for micro-encapsulation determines the physical andchemical properties of the resultantmicrospheres. Such polymers should be

chemically compatible, non-reactive with thecore material and provide the desired coatingproperties such as strength, flexibility,impermeability, optical properties and stability(1). The use of natural biodegradable polymersas coating materials in microsphereformulations has received considerableattention (1, 2). They remain attractive primarilybecause they are readily available, relativelyinexpensive, biodegradable and capable of alarge number of chemical modifications. A

*Corresponding author: Adenike Okunlola, Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria, Tel: 234 8023351064, E-mails: [email protected], [email protected]

This Journal is © IPEC-Americas Inc March 2016 J. Excipients and Food Chem. 7 (1) 2016 - 4 DOWNLOAD FREE FROM HTTP://OJS.ABO.FI/JEFCThis material MAY NOT be used for commercial purposessee Creative Commons Attribution NonCommercial‐NoDerivatives 4.0 International

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majority of investigations of natural polymersas matrices in drug delivery systems havecentered on proteins and polysaccharidesincluding starches (2). Unmodified nativestarches, irrespective of their source, areundesirable for many applications because oftheir inability to withstand processing such asextreme temperature, extreme pH, high shearrate or freeze/thaw variation. To overcomethis, modifications are usually carried out tointroduce desirable properties, to repressinherent properties that are undesirable or toimpart new properties to meet a requirementfor a specific application. The three commonapproaches for modifying starches arechemical, physical or enzymatic modification.

Chemical modification is generally achieved byetherification, esterification, crosslinking,oxidation, cationization or grafting of starches.In the acetylation reaction, the number of acetylgroups incorporated onto the starch molecule,and the rate and efficiency, depends on thereagent type and concentration, pH, presenceof catalyst, reaction time, botanical origin of thestarch and on the size and structuralcharacteristics of the native granule (3, 4). Theacetylation of starch results in a considerablymore hydrophobic material which has beenshown to have better retention of tensileproperties in aqueous environments (5). Whenstarch reacts with acid, it loses the hydroxylgroup and the free hydroxyl groups on C2, C3

and C6 of the starch molecule are substitutedwith acetyl groups. As the degree ofsubstitution (DS) increases, the nature of theacetylated starch changes to hydrophobic andinterparticular bonding capacity increases (6).They form flexible, water insoluble films thatare acid and heat-resistant and can substantiallyretard drug release (7).

Oryza glaberrima Steud (family Poaceae) alsoknown as Ofada rice is a particular delicacy inNigeria where it is largely cultivated andprocessed in the southwestern rice producing

communities (8). The rice grain’s high starchcontent makes it a less expensive source ofstarch which render it a cost effectivepharmaceutical excipient. The modification ofOfada rice starch by acetylation with varyingdegrees of substitution (DS 1.42; DS 2.62) wascarried out to produce polymers which werethen used to formulate nimesulide.

Nimesulide (4'-nitro-2'-phenoxy methanesulfonanilide) was introduced in 1985 as one ofa number of potent nonsteroidal anti-inflammatory drugs (NSAIDs) useful fortreating various inflammatory conditions (9). Itis a weakly acidic drug and differs from othernonsteroidal anti- inflammatory drugs(NSAIDs) in that its chemical structurecontains a sulfonanilide moiety, rather than acarboxylic group, as the acidic group. However,as with other non-steroidal anti-inflammatorydrugs, nimesulide is fairly insoluble in water (.0.01 mg/ml). This low aqueous solubility andwettability of the drug makes it morechallenging to formulate for oral or parenteralpharmaceutical dosage forms, which may resultin variable bioavailability. To overcome someof these drawbacks, in this study nimesulidewas encapsulated and formulated intomicrospheres using acetylated Ofada starchwith DS 1.42 and DS 2.62 for sustained drugrelease. The microspheres were prepared by thequasi-emulsion solvent diffusion method. A full23 factorial experimental design was alsoperformed using the degree of substitution ofstarch (X1), ratio of drug to polymer (X2) andpolymer concentration (X3) as the independentvariables. The effects of variation of thesefactors on some properties of nimesulidemicrospheres were then determined.

MATERIALS AND METHODS

Starch extraction and acetylation

Starch was extracted from the Ofada rice grainsaccording to a procedure previously reportedby Okunlola et. al., (10) and the modification


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was carried out by acetylation according to themethod by Singh and Nath, 2012 (11). Briefly,fifty grams of native starch was suspended in550 ml of de-ionized water in a 1000 ml conicalflask. The suspension was gelatinized by stirringat a temperature above 65EC, which is thegelatinization temperature of Ofada starch, for30 minutes on a hotplate (MS 400 Hot PlateMagnetic Stirrer, LIDA Instrument, ShanhgaiChina). The gelatinized starch was precipitatedusing 1 litre of anhydrous ethanol. Theprecipitated material was filtered and theresidue washed with acetone, filtered again anddried at 50°C (GallenKamp MoistureE x t r a c t i o n O v e n - M o d e l : B S 2 5 0 ,GallenKamp Co., UK)

The dried powder was screened (sieve size125 µm). Twentyfive grams of thepregelatinized starch was dispersed in200 grams of pyridine in a 1-liter round-bottomflask. One hundred grams of acetic anhydridewas added to the dispersion. The flask wasplaced into an oil bath and maintained at 100ECrotating at low speed inside a fume hood. Toprepare acetylated starches with DS 1.42 and2.62, the reaction was carried out for 3 and 4.5hours respectively with continuous stirring. Atthe end of the reaction, the mixture wastransferred to a beaker and cooled to roomtemperature and the product was precipitatedfrom 1300 ml of ethanol under high shearhomogenization. The precipitate was filtered,washed thoroughly with ethanol to remove theresidual pyridine in the precipitate and, thenfiltered again, before drying in an oven at 40EC(GallenKamp Moisture Extraction Oven-Model:BS 250 GallenKamp Co., UK) (12).

Determination of degree of substitution

One gram of acetylated starch and 50 ml of75% ethanol were mixed in a flask with a loosestopper. The mixture was stirred in a water bathat 50EC for 30 minutes. After cooling to roomtemperature, 40 ml of 0.5 N potassium

hydroxide (KOH) solution was added. Theflask was fitted with a tight stopper and kept atroom temperature for 72 hours shakingoccasionally for complete saponification. Theexcess of alkali in the solution was titrated with0.5 N HCl solution using phenolphthalein asthe indicator. A blank test was performedfollowing the same procedure. The percent ofacetyl group and degree of substitution (DS)were calculated using Equations 1 and 2 (13):

Eq. 1 Acetyl group (%) = V - V x M x 0.043 x 100

S

b s

w

where, Vb is the blank titration volume in ml, Vs

is the sample titration volume in ml, M is themolarity of HCl and Sw is the sample weight ingrams.

Eq. 2Degree of Substitution = 162 x % Acetyl group

4300 - (42 x % Acetyl group)

where, 162 is the molecular weight of theanhydroglucose unit, 42 is the molecular weightof the replaceable acetyl group and 4300 is themolecular weight of the acetyl groups attachedto 100 anhydroglucose units.

Morphology

The shape and size of the native and modifiedstarch granules were determined using ascanning electron microscope (Hitachi SU8030FE-SEM Tokyo, Japan) at an acceleratingpotential of 5.0 kV. All the samples weresputter-coated with Au/Pd prior toexamination.

Fourier Transform Infrared SpectroscopyAnalysis

Starch powder (1% of the KBr amount) wasmixed with the KBr powder and trituration wascarried out using an agate mortar for 5 minutes.The die-set was assembled and the powder-


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mixture was transferred into it. The powderwas pressed for 2 minutes (Thermo QwikHandi-Press P/N 0016-125) to form a thin andtransparent KBr disc. The starches wereanalyzed using a Fourier Transform InfraredSpectroscopy (FTIR) (FT-IR-Thermo NicoletNexus 870 Madison, WI, USA) in transmissionmode. Transmission spectra were recordedusing at least 64 scans with 8 cm-1 resolution inthe spectral range 4000–400 cm-1.

Preparation of the nimesulide microspheres

The nimesulide microspheres were preparedusing the quasi-emulsion solvent diffusionmethod of spherical crystallization. One gramof nimesulide and 2 grams of acetylated Ofadastarch were dissolved completely in 100 ml ofchloroform. In the drug-polymer mixture,1 gram of Aerosil was suspended uniformly byvigorous agitation. The resultant drug-polymer-Aerosil suspension was poured into distilledwater (750 ml) containing 0.08% of SodiumDodecyl Sulphate contained in a 2-liter beakerunder moderate agitation 450-750 RPM(Talboys Laboratory Stirrer Model No: 102USA) and thermally controlled at 38EC(Maplelab Scientific Stirrer-Hotplate SHC-11,UK). Using agitation the suspension wasimmediately dispersed into fine quasi-emulsiondroplets and the drug and polymers co-precipitated in the emulsion droplets. Afteragitating the system for 20 minutes, 750 ml ofSDS was added slowly to promote the diffusionof the chloroform from emulsion droplets intoSDS thereby enhancing the solidification of thequasi-emulsion droplets. Agitation wascontinued for another 40 minutes until thetranslucent quasi-emulsion droplets turned intoopaque microspheres. The solidifiedmicrospheres were recovered by filtration andwashed with water, and the resultant productswere dried in a Gallenkamp BS Oven 250 at50EC for 6 hours. This procedure producedmicrospheres with a varying ratio of drug topolymer and polymer concentrations (1:2 and1:4; 2 and 4 % w/v respectively). The variousbatches and their composition are presented inTable 1.

Table 1 Composition of nimesulide microsphereformulations

INGREDIENTS B1 B2 B3 B4 B5 B6 B7 B8

DRUG (g) 1.0 0.5 2.0 1.0 0.5 1.0 2.0 1.0

ACETYLATED STARCHDS 1.42 (g)

2.0 2.0 4.0 4.0 - - - -

ACETYLATED STARCHDS 2.62 (g)

- - - - 2.0 2.0 4.0 4.0

AEROSIL (g) 1.0 0.5 2.0 1.0 0.5 1.0 2.0 1.0

CHLOROFORM (ml) 100 100 100 100 100 100 100 100

0.08 %w/v SDS SOLUTION(ml)

1500 1500 1500 1500 1500 1500 1500 1500

DRUG:POLYMER 1:2 1:4 1:2 1:4 1:4 1:2 1:2 1:4

POLYMER CONC(% w/v)

2 2 4 4 2 2 4 4

Experimental design

A full 23 factorial experimental design wasperformed using three factors, each at twolevels. The eight possible combinations areshown in Table 2. The degree of substitutionDS (X1), ratio of drug to polymer (X2) andpolymer concentration (X3) were chosen asindependent variables while microsphere size,swelling, entrapment and time taken for 80%drug release (t80) were selected as dependentvariables. The main effects (X1, X2 and X3)represent the average result of changing onefactor at a time from its low to high value.

Characterization of Microspheres

Size and morphology

The morphology and surface characteristics ofthe microspheres were observed using aScanning Electron Microscope (HitachiSU8030 FE-SEM Tokyo, Japan) at anaccelerating potential of 5.0 kV. All the sampleswere sputter-coated with Osmium tetra oxideprior to examination using the Osmium plasmacoater (OPC 60 X1, Japan). The particle sizesof 100 microbeads were determined by opticalmicroscopy.

Swelling index

Exactly 1 ml of microsphere bed was soaked inphosphate buffer (pH 7.4) inside a 10 mlmeasuring cylinder for 12 hours and the swel-


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Table 2 Factorial Design for the formulation and evaluation of batches

BATCHCODE

CODEDLEVELS

REAL VALUESParticle

size (µm)

Swelling

(v/v)

Entrapment (%)

t80

(h)X1 X2 X3

X1

Degree ofsubstitution

X2

Drug : Polymer

X3

Polymerconcentration %

w/v

B1 -1 -1 -1 1.42 1:2 2 50.91 ± 16.22 1.35 ± 0.05 56.75 ± 0.45 2.00 ± 0.21

B2 -1 +1 -1 1.42 1:4 2 57.98 ± 20.15 1.30 ± 0.02 71.60 ± 2.36 3.10 ± 0.30

B3 -1 -1 +1 1.42 1:2 4 54.59 ± 19.67 1.42 ± 0.00 76.75 ± 0.45 3.40 ± 0.18

B4 -1 +1 +1 1.42 1:4 4 74.24 ± 24.73 1.20 ± 0.00 88.02 ± 3.01 4.60 ± 0.36

B5 +1 +1 -1 2.62 1:4 2 21.05 ± 4.25 1.15 ± 0.01 65.48 ± 4.80 5.00 ± 0.50

B6 +1 -1 -1 2.62 1:2 2 30.10 ± 5.33 1.10 ± 0.05 95.80 ± 2.58 5.40 ± 0.40

B7 +1 -1 +1 2.62 1:2 4 40.55 ± 3.85 1.22 ± 0.00 96.65 ± 0.07 7.00 ± 0.45

B8 +1 +1 +1 2.62 1:4 4 46.10 ± 3.85 1.20 ± 0.03 98.28 ± 2.30 7.10 ± 0.96

ling index was calculated as the ratio of thevolume to the original volume after 12 hours.

Entrapment Efficiency

A quantity of microspheres containing 50 mgof drug was accurately weighed and crushedusing a glass mortar and pestle and thensuspended in 50 ml of phosphate buffer, pH7.4. After 24 hours, the solution was filteredand the filtrate was analyzed for drug using aSpectrumlab 752s UV-VIS spectrophotometer(Ningbo Biocotek Scientific Instruments Co.,Ltd, Zheijang, China) at 295 nm. The drugentrapment efficiency (E) was calculated usingEquation 3:

Eq. 3 E (%) = Practical drug content

Theoretical drug content x 100

Drug release study

The in vitro dissolution studies were carried outusing the paddle method (USP XXI), rotated at50 RPM in 900 ml of phosphate buffer, pH 7.4,maintained at 37 ± 0.5EC. The microspheres(containing 100 mg drug) were placed in thedissolution medium. Samples (5 ml) werewithdrawn at different intervals and replacedwith equal amounts of fresh medium. Theamount of nimesulide released was determinedat wavelength of 295 nm, using the

Spectrumlab 752s UV-VIS spectrophotometer.Determinations were done in triplicate.

Modelling and comparison of release profiles

Data obtained from the in vitro release studieswere fitted to various kinetic equations todetermine the kinetics and mechanisms of drugrelease from the microspheres. The results ofthe drug release from the formulations werefitted to Zero Order, First Order, Higuchi (14),Hixon-Crowell (15) and Korsemeyer-Peppasmodels (16). The model of best fit wasidentified by comparing the values ofcorrelation coefficients.

RESULTS AND DISCUSSIONS

Characterization of starches

Acetylated Ofada starches with degrees ofsubstitution of 1.42 ± 0.02 and 2.62 ± 0.07were obtained. The acetyl content of themodified starches were 27.52 ± 0.23% and41.93 ± 0.90 %, respectively.

Scanning Electron Microscopy

The scanning electron micrographs are shownin Figure 1. Native Ofada starch in its nativeform had polyhedral granules with mean


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Figure 2 FTIR spectra for (a) native and (b) acetylatedOfada rice starches.

Figure 3 Scanning Electron Micrograph of nimesulidemicrospheres containing acetylated Ofada rice starch Mg x500.

Figure 1 Scanning electron micrographs of (a) nativeOfada rice starch and (b) acetylated Ofada rice starch Mgx 800.

particle sizes of 2.20 ± 0.05 μm. Acetylation ofstarch disrupted the granular structure of thenative starches and the acetylated starchesshowed significantly (p<0.01) larger, fibrous,irregular aggregates with a mean size of 17.80 ±0.75 μm.

FTIR analysis

The FTIR spectra of native and acetylatedstarches are presented in Figure 2. FTIRspectroscopic analysis showed that thecharacteristic absorption intensities of esterifiedstarch increased with increase in the degree ofsubstitution, and the characterized peak of

hydroxyl group almost disappeared in thespectrum of DS 2.62 acetylated starch. Newbands at 1700 cm-1 (Stretching C=O), 1375 cm-

1(Stretching C-CH3) were observed for theacetylated starches. FTIR bands at 3400cm-1

(Stretching O–H) and 1083cm-1 (C-O-C bondstretching) were weakened, confirming thereplacement of the hydroxyl groups in thestarch molecules with acetyl group (10, 17).

Characterization of microspheres

Microspheres of nimesulide were formulatedusing the acetylated Ofada rice starch aspolymers by the quasi-emulsion solventdiffusion method. Compared with the solventevaporation method for microspherepreparation, the solidification of the liquiddroplet in this process is much faster.Furthermore, the method did not require theuse of antiadhesion agents, such as talc, whichare often used in the evaporation method. Thesize of the micospheres, the entrapment, andrelease characteristics of drugs have beenreported to be dependent on the method usedto prepare the microspheres (18). The scanningelectron micrographs of the nimesulidemicrospheres containing the acetylated starch


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Figure 4 FTIR spectra of nimesulide, acetylated Ofada ricestarch and acetylated starch based nimesulide microspheres.

are shown in Figure 3. The microspheres werediscrete particles that were near-spherical inshape and no drug crystals were observed. Theparticle size analysis revealed that the meandiameter of the microspheres containing starch DS 1.42 was larger than that of DS 2.62.(50.91±16.22 to 74.24±24.73 µm and21.05±4.25 to 46.10±3.85 µm, respectively).Furthermore, as the concentration of starchincreased, the size of the microspheres alsoincreased. Formulations with higherconcentrations of starch produce viscouspolymer solutions which may be difficult tobreakdown into smaller droplets, thus resultingin larger microspheres. The observed increasein surface smoothness with decreasing polymerconcentration could be attributed to the rapid

diffusion of the solvents from the formulationshaving low polymer concentration (19). TheFTIR spectra shown in Figure 4 showed thatthe drug was well entrapped into the polymers.

The characteristic peaks for microspheres in theFTIR spectra was found to be super-imposableto that of the pure drug. There were no extrapeaks, band shift and broadening compared tothe pure drug and polymers, which showed thatthere was no drug polymer interaction.

Experimental design

The 23 full factorial experimental design wascarried out using Minitab 16 Statistical Software(Minitab Inc., USA). The experimental designprovides a clear indication of the quantitativeeffects of the three parameters, the degree ofsubstitution of starch polymer (X1), drug:polymer ratio (X2) and polymer concentration(X3) on particle size, swelling, entrapmentefficiency and dissolution time. This allows thethree factors to be varied simultaneously, thusenabling evaluation of the effects of eachvariable at each level as well as showing theinterrelationships among them (20). The codedand real values of the factors and responsevariables are presented in Table 2 while thevalues of the individual and interactioncoefficients are presented in Table 3.

Table 3 Summary of the individual and interaction coefficients of the variables on the particle size, swelling, entrapmentand dissolution time of nimesulide microspheres

FACTOR COEFFICIENTPARTICLE

SIZE(µm)

SWELLINGENTRAPMENT

(%)t80 (h)

X1Effect -12.49 -0.08 7.89 1.43

p-value 0.01 0.33 0.33 0.02

X2Effect 2.90 -0.03 0.32 0.25

p-value 0.06 0.25 0.95 0.13

X3Effect 6.93 0.02 8.76 0.85

p-value 0.02 0.40 0.30 0.04

X1 X2Effect -3.78 0.04 -6.85 0.33

p-value 0.04 0.21 0.37 0.10

X1X3Effect 1.94 0.03 -0.35 0.10

p-value 0.08 0.30 0.95 0.29

X2X3Effect 3.40 -0.03 3.55 0.08

p-value 0.05 0.25 0.57 0.37


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The relative magnitudes of the effects of thefactors on these variables can be observedeither as a positive effect which shows anincrease in magnitudes of the response value ora negative effect that shows a decrease.

Particle size

The size of microspheres strongly affects therate of drug release. As the size decreases, thesurface area-to-volume ratio of the particleincreases. Thus, for a given rate of drugdiffusion through the microsphere, the rate offlux of drug out of the microsphere, per massof formulation, will increase with decreasingparticle size (21).

The results show that the coefficient value wasnegative for the influence of DS (X1) onparticle size indicating that changing the degreeof substitution from 1.42 to 2.62 resulted in adecrease in the size of the microspheres. Thecoefficients that influence the drug:polymerratio (X2) and polymer concentration (X3) forparticle size were positive, indicating that anincrease in these factors resulted in increase inthe sizes of microspheres produced. Theranking of the coefficients for particle size was X1 >X3 >X2 showing that DS was the mostsignificant variable (p = 0.01) that influencedthe size of the formulated microspheres.

The values of the interaction coefficients forparticle size were X1X2 >X2X3>X1X3. Thisindicates that the interaction between DS anddrug:polymer ratio (X1X2) had the greatesteffect (p = 0.04) on particle size. The value ofthe interaction coefficient X1X2 was negativeindicating that both DS and drug:polymerpolymer ratios interacted to producemicrospheres of reduced size.

Swelling

The negative coefficient values of X1 and X2 onswelling indicated that changing the DS from1.42 to 2.62 and increasing the drug:polymer

ratio from 1:2 to 1:4 produced microsphereswith reduced swelling. The results also showeda positive value for X3 suggesting that anincrease in the polymer concentration increasedthe swelling of the nimesulide microspheres.This could be due to the high water sorptioncapacity of the starch indicating theeffectiveness of the Ofada starch to increaseswelling with increasing concentrations ofpolymer (22). The order of the coefficients forswelling was X1>X2>X3 suggesting that DSwas also the variable that influenced theswelling behavior of the microspheres the most.

The values of the interaction coefficients onswelling was X1X2 > X1X3 = X2X3, indicatingthat the interaction between DS and drug:polymer ratio (X1X2) had the greatest effect onthe swelling of the microspheres. The positivevalue of the interaction coefficient indicatesthat the variables interacted causing an increasein the swelling of the microspheres.

Entrapment

Entrapment efficiency for all the formulationsvaried from 56.75 ± 0.45 to 98.28 ± 2.30%,showing a dependence on the degree ofsubstitution of the starch, ratio of the drug:polymer, as well as, polymer concentration.Entrapment increased with increasing DS,polymer ratio and polymer concentrations (23,24). It was observed that B1-B4 containinglower DS showed lower values of entrapmentthan those for B5-B8, due to the relatively lesshydrophobic nature of the starch DS 1.42.

The coefficients value were positive for theinfluence of the three factors on entrapment,suggesting that entrapment efficiency increasedwith increase in these factors. The ranking ofthe coefficients on entrapment was X3>X1>X2

suggesting that the polymer concentration hadthe greatest effect on the entrapment of thedrug in the microspheres containing theacetylated starches. The ranking of the


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Figure 5 Dissolution profile of nimesulide microspheres.

interaction coefficients on entrapment wasX1X2>X2X3>X1X3. The interaction betweenDS and drug:polymer ratio (X1X2) had thegreatest effect on the percentage entrapment ofnimesulide in the microspheres.

Dissolution

The in vitro release of nimesulide from thestarch-based microspheres was carried out in apH 6.8 phosphate buffer media to simulate thephysiological condition. The dissolution profilefor all the batches are shown in Figure 5. Thepercentage cumulative release was lower for theB5-B8 containing starch DS 2.62 than for the B1-B4 containing starch DS 1.42. Microspherescontaining higher polymer ratio and higherpolymer concentration showed a similar trendof cumulative release.

The coefficient values of X1, X2 and X3 on thedissolution time (t80) were positive indicating

the increase in the three factors that producedmicrospheres with longer dissolution times.The ranking of the coefficients on t80 was X1>X3>X2 indicating that the DS had the greatesteffect (p = 0.02) on the time of the release ofnimesulide from the microspheres. The valuesof the interaction coefficients for t80 wereranked as X1X2 >X1X3 >X2X3 showing that DSand drug:polymer ratio (X1X2) interactedsynergistically to prolong the time taken for therelease of nimesulide from the microspheres.

Contour plots were developed by coding thevalues of the degree of substitution and drug:polymer ratio and are shown in Figure 6. Acontour plot is a graphical representation of therelationships among three numeric variables intwo dimensions (25). The contour plots can beused to predict the relevant responses as theyreveal the relative effects of the variables on theresponses. Figures 6 (a), (b), (c) and (d) showthe influence of the DS and the drug:polymer


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Figure 6 Contour plots of (a) size, (b) swelling, (c) entrapment and (d) dissolution time (t80)if nimesulide microspheres as a function of the degree of substitution of starch and theconcentration of starch.

ratio on the size, swelling, entrapment and t80

respectively. The darker region of the plotsshows that the degree of substitution exerted ahigher effect on the entrapment and dissolutiontime than drug:polymer ratio in the acetylatedOfada starch-based nimesulide microsphereformulations. The contour plots show that theacetylated Ofada rice starch with DS 2.62 wassuitable for the microsphere formulationsbecause they were capable of high entrapmentefficiency and sustained release.

Drug Release Kinetics and Models

Drug release from polymeric microspheres canoccur in 3 phases. First, through rapid release(burst release) that is attributed to drug that isadhered to the wall of the microspheres.Second, through slow release, which dependson the degradation kinetics of the polymer usedin the microspheres and third throughaccelerated delayed release, which depends onthe diameter of the microsphere (18, 19). Thetwo main factors that influence drug release


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from polymeric microspheres are thedegradation of the polymer matrix anddiffusion, both of which are influenced by thepolymer morphology (18).

Acetylated Ofada rice starch used in this studyis an amorphous and biodegradable material inwhich acetylation of the starch reduces itsenzymatic degradation, thereby improving itspotential as a carrier system. Thebiodegradation of acetylated starch is known tobe inversely proportional to its degree ofsubstitution (26). At the initial stage of drugrelease from the starch-based microspheres,nimesulide loosely bound on the surface of themicrospheres is released by a mechanism ofdiffusion through the surface due to wateruptake immediately after exposure. At the laterstage, the drug releases more slowly, the ratebeing determined by the diffusion through thepolymer matrix. The diffusion of thenimesulide occurs through the amorphousregion of the starch polymer. Simulating thedrug kinetics using different models (Zeroorder, First order, Higuchi, Hixon-Crowell andKorsemeyer-Peppas) as presented in Table 4, itwas observed that generally, drug release for themicrospheres containing acetylated Ofada ricestarch with DS 1.42 (Formulations B1, B2 andB3) fitted the First Order kinetic model with theexception of B4 which fitted the Korsmeyer-Peppas model (n= 0.3997).

Table 4 Correlation coefficients obtained for release ofnimesulide from microspheres using different kineticmodels (n = 3)

FORMULATIONZERO

ORDERFIRST

ORDERHIGUCHI

HIXSON-CROWELL

KORSMEYER

R2 n

B1 0.6461 0.9785 0.8768 0.8722 0.8472 0.3116

B2 0.6786 0.9296 0.8990 0.9258 0.9203 0.3187

B3 0.7547 0.9812 0.9389 0.9356 0.9152 0.4048

B4 0.8407 0.9176 0.9783 0.9783 0.9823 0.3997

B5 0.9130 0.9644 0.9829 0.9846 0.9652 0.6446

B6 0.9363 0.9718 0.9793 0.9905 0.9775 0.2727

B7 0.9936 0.9006 0.9540 0.9848 0.9975 0.7727

B8 0.9961 0.8846 0.9315 0.9821 0.9964 0.8716

In a First Order model, drug release isdependent on the remaining concentration of

drug in the microspheres. The microspherescontaining acetylated Ofada rice starch withthe DS 2.62 in B5 and B6 fitted the Higuchimodels as indicated by their correlationcoefficients. Higuchi developed theoreticalmodels to study the release of highly andminimally water-soluble drugs incorporated inan insoluble matrix. According to this model,drug release was a diffusion process that wasdependent on the square root of time. Higuchiproposed this model of drug release based onthe assumptions that the initial concentration ofdrug in the matrix is much higher than the drugsolubility; matrix swelling and dissolution arenegligible (14). Formulations B7 and B8 showedsimilar drug release kinetics to B4 i.e., theKorsmeyer-Peppas model (n = 0.7727 and0.8716 respectively). In this model, the value ofn characterizes the release mechanism of thedrug. When 0.45 # n, this corresponds to aFickian mechanism, 0.45 < n < 0.89 to nonFickian transport, n = 0.89 to case II(relaxational transport) and n > 0.89 to supercase II transport (16, 27). From the values ofthe slopes, the drug release mechanism fromthe formulation B4 containing the acetylatedOfada starch DS 1.42 is considered Fickianwhile those of B7 and B8 are non-Fickian.

CONCLUSION

Acetylated Ofada starches with degrees ofsubstitution of 1.42 ± 0.02 and 2.62 ± 0.07were used in the formulation of nimesulidemicrospheres at varying drug:polymer ratiosand polymer concentrations. Microspherescontaining acetylated Ofada rice starch DS 2.62showed significantly smaller particle size,reduced swelling, higher entrapment and longerdissolution times. The degree of substitutionwas the most significant variable that influencedthe size, swelling and dissolution time ofnimesulide microspheres while polymerconcentration had the most influence onentrapment. The drug release fitted mainly intothe Korsmeyer kinetic models, as well as, First


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Order and Higuchi kinetic models. This studysuggests the usefulness of acetylated Ofada ricestarches with the DS 2.62 for the formulationof microspheres with high entrapment andprolonged drug release which can serve as asuitable substitute to synthetic polymers in drugdelivery.

DECLARATION OF CONFLICT OF INTEREST

The authors hereby declare that there is noconflict of interest.

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