instantaneous enteric nano-encapsulation of omeprazole: pharmaceutical and pharmacological...

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International Journal of Pharmaceutics xxx (2014) xxx–xxx

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Pharmaceutical nanotechology

Instantaneous enteric nano-encapsulation of omeprazole:Pharmaceutical and pharmacological evaluation

Ehab R. Bendas *, Aly A. AbdelbaryDepartment of Pharmaceutics, Faculty of Pharmacy, Cairo University, Kasr El-Ainy Street, Cairo 11562, Egypt

A R T I C L E I N F O

Article history:Received 11 February 2014Received in revised form 10 April 2014Accepted 14 April 2014Available online xxx

Keywords:Enteric nanocapsulesOmeprazoleHydroxypropyl methylcellulose phthalateIn vivo antiulcer activity

A B S T R A C T

Recently, great attention has been paid to nanocapsules. The interest of these structures is due to theirpromising applications as drug delivery systems. The objective of this study was to develop novel entericcoating technique based on instantaneous encapsulation of the acid-labile drug, omeprazole ininnovative enteric nanocapsules. Omeprazole enteric nanocapsules were formulated by varying the typeand amount of the enteric polymer. The particle size (PS), polydispersity index (PDI), zeta potential (ZP)and encapsulation efficiency (EE) values of the prepared enteric nanocapsules were determined. A full21�31 factorial design was used for planning and analysis of the experimental trials to select theoptimized formulation. The highest desirability value was 0.7463 for formula E3 (containing 200 mghydroxypropyl methylcellulose phthalate (HPMCP)). The stability of omeprazole was reflected by theabsence of the exothermal peak when the drug was encapsulated as detected by differential scanningcalorimetry (DSC) thermograms. In vitro drug release study confirmed the USP specifications required tomeet the key formulation characteristics of gastro-resistance. In vivo pharmacological assessmentshowed that the optimized nanocapsules were able to protect rat stomach against ulcer formationcompared to the aqueous suspension of the drug which showed less significant protection.

ã 2014 Published by Elsevier B.V.

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsev ier .com/locate / i jpharm

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1. Introduction

Research in nanotechnology has powerfully increased in thelast decades. Many colloidal carriers, such as liposomes(Gregoriadis, 1995), niosomes (Rajera et al., 2011), nanoemulsions(Tiwari and Amiji, 2006) and nanoparticles (Agnihotri et al., 2004)have been extensively investigated for drug delivery.

The main advantages of nanoparticles are biocompatibility andbiodegradability, control of the drug release, increase of drugselectivity and effectiveness, improvement of drug bioavailabilityand decrease of drug toxicity and the ability to target specifictissues (Malam et al., 2009). Polymeric nanoparticles are namednanocapsules, when they contain a polymeric wall and an oil core(Jager et al., 2007), where the core acts as a liquid reservoir forseveral molecules or drugs, and the wall as a protective membrane.They have special use for encapsulating and delivering hydropho-bic drugs (Sanchez-Moreno et al., 2012). The versatility of thesenanocapsules for an efficient encapsulation in their oily core ofseveral anti-cancer drugs has been previously demonstrated(Beduneau et al., 2007).

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48* Corresponding author. Tel.: +20 225311260/22835173; fax: +20 223628426.E-mail address: [email protected] (E.R. Bendas).

http://dx.doi.org/10.1016/j.ijpharm.2014.04.0300378-5173/ã 2014 Published by Elsevier B.V.

Please cite this article in press as: Bendas, E.R., Abdelbary, A.A., Instantanepharmacological evaluation, Int J Pharmaceut (2014), http://dx.doi.org/1

Omeprazole, a substituted benzimidazole, is a powerfulinhibitor of gastric acid secretion in man probably without adirect effect on pepsin secretion (Festen et al., 1986). It acts by non-competitive inhibition of parietal cell H+/K+-ATPase (Felleniuset al., 1981). Omeprazole has a half-life of less than 1 h and isalmost entirely cleared from plasma within 3–4 h. Omeprazole ismetabolized in the liver. The two major plasma metabolites aresulphone and hydroxyomeprazole, neither of which contributes tothe antisecretory activity (Cederberg et al., 1989). Omeprazoleseems to be well absorbed from the gastrointestinal tract (GIT).However, its oral bioavailability in humans is about 40–50% due toa marked first-pass metabolism before entering the systemiccirculation (Watanabe et al., 1994). Omeprazole is widely used indoses of 20–80 mg in treatment of duodenal and gastric ulcers,reflux oesophagitis and in the Zollinger–Ellison Syndrome (Clissoldand Campoli-Richards, 1986; Watanabe et al., 1994).

Omeprazole is sensitive to heat, humidity, light, and organicsolvents (Turkoglu et al., 2004). It degrades rapidly in acidsolutions (Quercia et al., 1997). To prevent acid degradation instomach, the drug is formulated as enteric-coated formulationssuch as enteric coated granules (Andersson et al., 1991) andenteric-coated tablets and capsules (Thomson et al., 1997).Differences in the quality of the granules coating are a potentiallimiting factor of the in vivo performance of the tested

ous enteric nano-encapsulation of omeprazole: Pharmaceutical and0.1016/j.ijpharm.2014.04.030

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Table 1Full factorial design for optimization of the enteric nanocapsules formulae.

Factors (independent variables) Levels

X1: type of polymer HPMCP PVAPX2: amount of polymer (mg) 50 100 200

Responses (dependent variables) ConstraintsY1: particle size (nm) MinimizeY2: polydispersity index MinimizeY3: zeta potential (mV) Maximize (least negativity)Y4: encapsulation efficiency (%) Maximize

2 E.R. Bendas, A.A. Abdelbary / International Journal of Pharmaceutics xxx (2014) xxx–xxx

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rmulations. It was found that omeprazole degradation is moreonounced in aqueous polymer dispersions than in organiclymer solutions (Riedel and Leopold, 2005). The influence ofganic polymer solutions on the stability of omeprazole depends

the amount of acidic groups in the polymeric structure, wherease influence of aqueous polymer dispersions depends on the pHlue of the dispersion. However, there are several concerns overe use of organic solvents, which usually are toxic, flammable, andplosive in addition to, the ecological and the economic problemsafati et al., 2006). Also there are still some potential problemssociated with the use of the aqueous dispersions in pharmaceu-al coating, comprising microbial contamination, the instability

the dispersions or the active ingredient, long processing timed adjusting optimal processing conditions. These criticalrameters can cause a non-uniformity of the applied coatingyer and up-scaling problems (Mehuys et al., 2005). To overcomee limitations of aqueous coating and to reduce process time, ay coating method was developed (Obara et al., 1999).The aim of this study was to develop novel enteric coating

chnique based on instantaneous encapsulation of the chemicallybile drug, omeprazole in innovative enteric nanocapsules andd optimal production parameters for a stable, highly concen-ated omeprazole formulation. Moreover, the pharmacologicalfects of the omeprazole enteric nanocapsules were evaluated by

in vivo antiulcer study using Wistar male rats.

Materials

Omeprazole was kindly provided by Aventis Pharma, Cairo,ypt. Lecithin and Pluronic F68 were purchased from Sigmaemical Co. (St. Louis, MO, USA). Hydroxypropyl methylcellulosethalate (HPMCP) was purchased from Samsung Fine Chemicals. (Seoul, Korea). Polyvinyl acetate phthalate (PVAP) was obtainedm Colorcon (West Point, PA, USA). Miglyol1 812 was supplied bysol Germany GmbH (Germany). Sodium bicarbonate wastained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).solute ethanol, acetone and acetonitrile (HPLC grade) wererchased from Fisher Scientific Co. (Pittsburgh, PA, USA). All otheremicals were of analytical grade.

Methods

. Preparation of omeprazole enteric nanocapsules

Omeprazole enteric nanocapsules were prepared according toe following procedures: 20 mg of omeprazole, 50 mg of lecithind different amounts of enteric polymers (HPMCP or PVAP) weressolved into a mixture of 2.5 mL of ethanol and 12.0 mL ofetone and then 315 mL of Miglyol1 812 was added. Immediatelyter that, this organic phase was poured into 25 mL of aqueouslution containing 62.5 mg of Pluronic F68 and 50.0 mg of sodiumcarbonate. Instantly upon addition of the organic phase into theueous solution, it turned milky. The milky mixture wasaporated under reduced pressure, in a rotary evaporatorotavapor, Type R 110, Büchi, Switzerland) at 45 �C for 15 min

a final volume of 20 mL. The nanocapsules formulae containingMCP were coded (E1–E3) while those containing PVAP wereded (E4–E6).

2. Characterization of enteric nanocapsules

2.1. Particle size and zeta potentialThe particle size of omeprazole enteric nanocapsules was

easured by photon correlation spectroscopy (PCS) using a

Please cite this article in press as: Bendas, E.R., Abdelbary, A.A., Instantanpharmacological evaluation, Int J Pharmaceut (2014), http://dx.doi.org

Zetasizer Nano ZS-90 instrument (Malvern Instruments, Worces-tershire, UK) after suitable dilution with distilled water. Themeasurements were conducted in triplicate. The average size ofthe nanoparticles was expressed as a median diameter (Dv50),which is particle diameter at 50% cumulative volume. Thepolydispersity index was also determined. The particles surfacecharge was quantified as zeta potential (z) using a Zetasizer NanoZS-90 instrument (Malvern Instruments, Worcestershire, UK).Measurements were also performed after suitable dilution withdistilled water and were conducted in triplicate.

3.2.2. Drug encapsulation efficiency (EE)The encapsulation efficiency (EE) of omeprazole was deter-

mined indirectly by calculating the difference between the totalamount of omeprazole added in the formulation and thatremaining in the aqueous medium after separating the entericnanocapsules by ultracentrifugation at 14,000 rpm for 1 h at 4 �Cusing cooling ultracentrifuge (Model 8880, Centurion ScientificLtd., W. Sussex, UK). Omeprazole contents were determined by anaccurate validated HPLC method. Encapsulation efficiency wascalculated using the equation:

EE ¼ Drug content in nanocapsulesDrug amount used

� 100 (1)

3.3. HPLC determination of omeprazole

An isocratic HPLC method was employed for the quantificationof omeprazole (Nataraj et al., 2012). A Thermo Separation HPLCsystem (Fremont, California) equipped with a P4000 pump unit, anAS3000 autosampler including an injection valve with a sampleloop of 50 mL volume, and a UV2000 detector was used. A ZorbaxExtend-C18 column (4.6 mm � 250 mm) containing 3.5 mm sizeadsorbent as stationary phase (Agilent Technologies, Santa Clara,California) was used. The column was maintained at roomtemperature (25.0 � 2.0 �C). The mobile phase consisted of amixture of phosphate buffer (pH 7.4) and acetonitrile (60:40 v/v).The flow rate and the UV detector were set at 1.0 mL/min and302 nm, respectively. Omeprazole was eluted at 6.3 min underthese conditions. An external calibration curve was established inthe range of 10–70 mg/mL. The assay procedures were validated interms of linearity, precision, and accuracy.

3.4. Optimization using statistical design

For systematic optimization of enteric nanocapsules formulae,full factorial experimental design methodology was employedwith Design-Expert1 software (Version 7, Stat-Ease Inc., MN) toinvestigate the effect of formulation variables on enteric nano-capsules properties (Table 1). Two independent variables wereevaluated which were: type (X1) and amount (X2) of the polymer.The particle size (Y1: PS), polydispersity index (Y2: PDI), zetapotential (Y3: ZP) and encapsulation efficiency (Y4: EE) were

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E.R. Bendas, A.A. Abdelbary / International Journal of Pharmaceutics xxx (2014) xxx–xxx 3

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selected as the dependent variables. Desirability was calculated forselection of the optimized formula which was subjected for furtherinvestigations.

3.5. Freeze drying of enteric nanocapsules

In order to study the in vitro dissolution and the physicalcharacters of the optimized formulation, the omeprazole entericnanocapsules formula (E3) was lyophilized after preparation. Thelyophilization was done after separation of the enteric nano-capsules by ultracentrifugation at 14,000 rpm for 1 h at 4 �C. Theconditions applied during this step were: freezing for 12 h at�20 �C and sublimation at �15 �C and 100 mbar for 12 h using aFlexi-DryTM MP Freeze Dryer (SP Scientific, Stone Ridge, NY), andfinally the secondary drying was carried out at 25 �C and 50 mbarfor 6 h.

3.6. Transmission electron microscopy (TEM)

The particle morphology of the optimized enteric nanocapsulesformula (E3) was examined using TEM (H-600, Hitachi, Japan). Thesample was dropped on copper–gold carbon grid and allowed todry. The grid was then mounted in the instrument and photo-graphs were taken at different magnifications (Thakkar et al.,2011).

3.7. Differential scanning calorimetry (DSC)

Thermograms of optimized omeprazole enteric nanocapsulesformula (E3), HPMCP, Pluronic F68, sodium bicarbonate andomeprazole were recorded on a DSC-60 (Shimadzu, Kyoto, Japan).Samples (5 mg) were placed in aluminum pans, and the lids werecrimped using a Shimadzu crimper. Thermal behavior of thesamples was investigated at a scanning rate of 10 �C/min andcovering temperature range of 20–300 �C. The instrument wascalibrated with an indium standard.

3.8. In vitro drug release

Drug-release study was implemented to confirm the USPspecifications required to meet the key formulation characteristicsof gastro-resistance. Amounts equivalent to 20 mg of omeprazole(E3) were tested using dissolution apparatus type II. Initially, theenteric nanocapsules were exposed to a simulated gastric fluid(500 mL of 0.1 N HCl) maintained at 37 � 0.5 �C and rotated at100 rpm for 2 h. After withdrawal of a 2 mL sample, 400 mL of0.235 M dibasic sodium phosphate at 37 �C were addedtothe 500 mLof 0.1 N hydrochloric acid medium in the vessel. The medium wasadjusted, if necessary, with either 2 N hydrochloric acid or 2 Nsodium hydroxide till a pH of 6.8 � 0.05 was reached. The rotationwas set at 100 rpm for another 1 h. 2 mL samples were withdrawn at10, 20, 30, 45 and 60 min and transferred to tubes containing 1 mL of0.3 N NaOH (El-Sayed et al., 2007). Samples were then filtered

Table 2Animal grouping and allocation of treatments for each group.

Groups

Control

Treatment 1

Treatment 2


through 0.1 mm PTFE syringe filter (Whatman Inc., Clifton, NJ, USA)and analyzed according to HPLC procedures described earlier.

3.9. In vivo antiulcer activity

The in vivo antiulcer activity of the optimized omeprazoleenteric nanocapsules formula (E3) was carried out using Wistarmale rats. The protocol for in vivo antiulcer activity was approvedby the ethical committee of Faculty of Pharmacy, Cairo University.Twenty-five male Wistar rats, weighing 150–200 g, were involvedin the study. The animals were supplied with standard diet and tapwater ad libitum. The rats were fasted for 24 h before theexperiments but had free access to water. During the fastingperiod, the animals were placed individually in cages with widemesh wire bottoms to prevent coprophagy. On the day ofexperiment, the rats were randomly divided into 3 groups with8 animals each as presented in Table 2. One rat was kept normal (noulcer induction and no treatment). Ulcers were induced in the 3groups by the oral administration of absolute ethanol (5 mL/kg)(Süleyman et al., 2002; Raffin et al., 2007; Gupta et al., 2012) to 24 hfasted Wistar male rats. The treatments (treatment 1 and 2) (20 mgof omeprazole/kg (Segawa et al., 1987; Amaral et al., 2013)) wereadministered orally by gavage feeding 1 h before the administra-tion of ethanol. After 1 h of the administration of ethanol, animalswere sacrificed by decapitation and the stomachs were removedand opened from the greater curvature region, and were washedwith normal saline and examined with the help of 3 foldmagnification for measurement of the lesion size. The destructionof animal carcasses was achieved by incineration. Ulcer index (UI)and percentage of ulcer protection were calculated according tothe following equations:

UI ¼ 10X

(2)

where, X = total mucosal area divided by the total ulceratedarea.

Percentage of ulcer protection ¼ UI of control � UI of testUI of control

� 100

(3)

4. Results and discussion

4.1. Formulation of omeprazole enteric nanocapsules

The innovative enteric coating technique hypothesized thatomeprazole can be encapsulated using Miglyol1 810 as an oilycore which is emulsified and stabilized by lecithin. Pluronic F68acts as surfactant to form the nanocapsule shell (Santander-Ortega et al., 2010) and the enteric polymers can be instan-taneously deposited on the nanocapsules to produce entericcoating. In case of drugs with major stability problems likeomeprazole, time of preparation is an important factor affectingits stability. The least amount of sodium bicarbonate (50.0 mg)was added in the aqueous phase to maintain the stability ofomeprazole during preparation.

Administered preparations

Ulcer induced (oral administration of water)Ulcer induced (oral administration of omeprazole suspension (20 mg/kg)Ulcer induced (oral administration of omeprazole formulation (20 mg/kg)



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Table 3Measured responses data used in the analysis of the factorial design.

Formulae X1: type of polymer X2: amount of polymer (mg) Y1: PS (nm) Y2: PDI Y3: ZP (mV) Y4: EE (%)

E1 HPMCP 50 208.7 � 10.8 0.66 � 0.07 �80.10 � 1.65 48.52 � 0.56E2 100 264.4 � 6.2 0.37 � 0.01 �79.10 � 0.95 49.59 � 0.81E3 200 246.3 � 4.4 0.18 � 0.01 �62.00 � 2.00 64.98 � 0.62E4 PVAP 50 433.0 � 12.7 0.74 � 0.03 �83.10 � 1.59 31.25 � 0.74E5 100 429.8 � 7.2 0.52 � 0.03 �67.10 � 0.95 37.28 � 0.86E6 200 494.7 � 7.5 0.35 � 0.01 �76.70 � 1.70 53.40 � 1.40

Figof


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2. Particle size, polydispersity index, zeta potential andcapsulation efficiency

The particle size, polydispersity index, zeta potential andcapsulation efficiency values of the prepared enteric nano-psules formulae are shown in Table 3. The prepared entericated nanocapsules formulae had particle size values rangingm 208.7 nm to 494.7 nm. The effects of type (X1) and amount2) of polymer on the particle size are shown in Fig. 1a. Factorialalysis of variance (ANOVA) showed a significant effect for type oflymer (X1) on the particle size (p = 0.0132). The nanocapsulesrmulae containing HPMCP showed lower particle size comparedith those containing PVAP. On the other hand, the particle size ofe prepared nanocapsules formulae was not affected by theount of polymer (X2) (p = 0.4249).In addition, both the type (X1) and amount (X2) of polymer had anificant effect on the polydispersity index (p = 0.0400 and

.1. Response 3D plots for the effect of type (X1) and amount (X2) of polymer on the paomeprazole enteric nanocapsules formulae.


p = 0.0123, respectively) (Fig. 1b). The polydispersity index wassignificantly decreased by increasing the amount of polymer.Moreover, lower polydispersity index values were observed in caseof nanocapsules containing HPMCP compared to the nanocapsulescontaining PVAP as shown in Fig. 1b.

When the thickness of adsorbed polymer layer increases, thezeta potential decreases but the nanocapsules are still stabilizeddue to steric effect. The decrease in zeta potential in terms ofabsolute value may be elucidated by the increase of thickness ofadsorbed polymer layer. This is due to the outward shift of theslipping plane, at which zeta potential is measured. In accordanceto Gouy–Chapman theory (Bolt, 1955), the slipping plane is movedto a point farther from the surface where the charge densitybecomes much smaller resulting in lower zeta potential(Abdelbary et al., 2013). It was found that the zeta potential ofthe prepared nanocapsules formulae was affected by neither thetype (X1) nor the amount of polymer (X2).

rticle size (a), polydispersity index (b), encapsulation efficiency (c) and desirability (d)



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Table 4Output data of the full factorial analysis of the enteric nanocapsules formulae.

Responses PS (nm) PDI ZP (mV) EE (%)

Minimum 208.7 � 10.8 0.18 � 0.01 �83.10 � 1.59 31.25 � 0.74Maximum 494.7 � 7.5 0.74 � 0.03 �62.00 � 2.00 64.98 � 0.62Ratio 2.37 4.11 0.75 2.08Transformation None None None NoneModel Linear Linear Mean LinearAnalysis Polynomial Polynomial Polynomial PolynomialAdequate precision 10.64 20.30 – 18.48Adjusted R2 0.94 0.97 0.00 0.97Predicted R2 0.77 0.90 �0.44 0.88Significant factors X1 X1 and X2 – X1 and X2

Fig. 2. Transmission electron micrograph of the optimized enteric nanocapsulesformula E3.


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The prepared enteric coated nanocapsules formulae hadencapsulation efficiency values ranging from 31.25% to 64.98%.The effects of type (X1) and amount (X2) of polymer on theencapsulation efficiency are demonstrated in Fig. 1c. The analysisof variance (ANOVA) showed a significant effect for both the typeand amount of polymer on the encapsulation efficiency (p = 0.0166and p = 0.0222, respectively). The encapsulation efficiency in-creased as the amount of polymer increased. Furthermore, asignificant increase in encapsulation efficiency was observedwhen HPMCP was the coating polymer compared to PVAP. Thiscould be due to the higher molecular weight of HPMCP (60,200)compared to PVAP (47,000) which resulted in higher encapsula-tion efficiency. Similar results were observed by Gaspar et al.working on L-asparaginase-loaded PLG nanoparticles (Gasperet al., 1998).

4.3. Analysis of factorial design

The factorial design was used for planning and analysis ofexperimental trials. The used design was a full 21�31 factorialdesign with statistical analysis through Design-Expert1 software.Adequate precision measured the signal to noise ratio to ensurethat the model can be used to navigate the design space (de Limaet al., 2011). A ratio greater than 4 (the desirable value) wasobserved in all responses except zeta potential as shown in Table 4.On the other hand, predicted R2was calculated as a measure of howgood the model predicts a response value (Chauhan and Gupta,2004; Kaushik et al., 2006). The adjusted R2 and predicted R2

should be within approximately 0.20 of each other to be in areasonable agreement (Annadurai et al., 2008). If they are not,there might be a problem with either the data or the model. It isworthy to note that the predicted R2 values were in a reasonableagreement with the adjusted R2 in all responses except zetapotential. This is due to that the zeta potential of the nanocapsulesformulae, as previously mentioned was affected by neither thetype nor the amount of polymer. Moreover, the model chosen forthe analysis of the zeta potential values was the mean not thelinear model.

For the selection of the optimum formula, it was almostimpossible to achieve all the desired responses simultaneouslybecause interference may occur (Singh et al., 2012). The optimumcondition reached in one response may have an opposite impacton another response. The desirability function combines all theresponses into one variable to predict the optimum levels for thestudied factors. Hence, desirability was calculated to select theoptimized formulae with the least particle size and polydispersityindex values, highest zeta potential (least negativity) andencapsulation efficiency values. The highest desirability valuewas 0.7463 for formula E3 (containing 200 mg HPMCP) as shownin Fig. 1d. Hence, this formula was selected for furtherinvestigations.


4.4. Evaluation of the freeze dried optimized formula (E3)

The freeze dried enteric nanocapsules formula (E3) was easilydispersed in distilled water. The particle size, polydispersity indexand zeta potential were evaluated after reconstitution and found tobe 251.5 � 3.4 nm, 0.170 � 0.01 and �65.22 � 1.52 mV, respectively.There was no significant difference in the measured parametersbefore and after freeze drying (p � 0.05). In addition, there was noleakage of the encapsulated drug after reconstitution as evidencedby absence of free drug in the dispersion.

4.5. Transmission electron microscopy (TEM)

The core–shell morphology of the nanocapsules containingoil cores was observed under the TEM, as shown in Fig. 2. Thenanocapsules are hollow and spherical. They revealed a smoothsurface, and the cores appeared white to gray. Similar resultswere obtained by Liu et al., working on polystyrene nano-capsules (Liu et al., 2011). In addition, the particle size shown bythe TEM micrograph was in a good agreement with that obtainedby PCS.

4.6. Differential scanning calorimetry (DSC)

DSC was performed to explore the physical changes thatoccurred in the drug after encapsulation into the enteric nano-capsules. It was realized from Fig. 3 that omeprazole showed a



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Fig. 3. DSC thermograms of pure omeprazole, NaHCO3, HPMCP, Pluronic F68, lecithin and the enteric nanocapsules formula (E3).


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ear endothermal transition at 156 �C followed by an exothermalansition at 164 �C. This behavior may reflect recrystallization ofe drug (Ruiz et al., 1998). Lecithin, Pluronic F68, NaHCO3 andMCP showed their peaks at 42, 51.9, 160.9 and 168.8 �C,spectively. The drug characteristic peaks disappeared in theermograms of the enteric nanocapsules formula (E3). This could

attributed to that omeprazole was entrapped in an amorphous molecular dispersion state in the polymeric enteric nanocapsuleith no further recrystallization (Ruiz et al., 1998; Dillen et al.,04; Sant et al., 2005).

7. In vitro drug release

Fig. 4 shows the release of omeprazole from enteric nano-psules formula (E3) that was first subjected to 2 h in 0.1 N HClllowed by 1 h at pH 6.8. Generally, omeprazole formulationsould be enteric coated. This is attributed to the fact thateprazole is sensitive to acidic conditions and after contact with

acid; omeprazole will degrade and will not function in its

Fig. 4. In vitro release profile of omeprazole from enteric nanocap


intended manner (He et al., 2009). The most important property ofan enteric coated dosage form is its resistance against gastricconditions (He et al., 2010; Missaghi et al., 2010). It is required thatno more than 10% drug degradation would occur after 2 h in 0.1 NHCl solution and not less than 80% omeprazole dissolve after45 min in alkaline pH (He et al., 2009). The enteric nanocapsulesformula (E3) complied with these conditions. Omeprazole was notreleased in the acidic conditions and the nanocapsules resisted theacidic medium. Visual observation of the nanocapsules formula(E3) after 2 h in the acid medium showed no signs of omeprazoledegradation. No yellow discoloration of the nanocapsules formulaor dissolution media appeared. Furthermore, the enteric nano-capsules formula released more than 80% omeprazole after 45 minin alkaline medium, pH 6.8.

4.8. In vivo antiulcer activity

The induction of ulcers by absolute ethanol is considered as agood model to evaluate the effect of omeprazole since it has been

sules formula (E3) at pH 6.8 after exposure to 0.1 N HCl for 2 h.



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Fig. 5. Photographs Q4of non-ulcer induced rat stomach (a) as compoared to opened rat stomachs after administration of absolute ethanol (b), omeprazole suspension (c),omeprazole enteric nanocapsules formula E3 (d).


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reported earlier (Wong et al., 1987; Chandranath et al., 2002). Oraladministration of absolute ethanol to the control groups clearlyshowed hemorrhagic lesions developed in the glandular portionof the stomach (Fig. 5). The in vivo evaluation showed that ulcerindex values were 5.69 � 1.14 for the control group, 4.24 � 0.95 fortreatment 1 (omeprazole suspension) and 0.79 � 0.39 fortreatment 2 (omeprazole enteric coated nanocapsules) (Table 5).One way analysis of variance (ANOVA) test showed statisticaldifferences (p � 0.05) between the ulcer index values. Post hocanalysis performed by SPSS1 software, version 9 (SPSS Inc.,Chicago, USA) utilizing LSD test showed that the omeprazoleenteric coated nanocapsules had a gastric ulcer index statisticallylower than both the omeprazole suspension and the control(p � 0.05). The percentages of ulcer protection were 25.61 and86.10% after the administration of treatment 1 (omeprazolesuspension) and treatment 2 (enteric coated nanocapsules),respectively. Alcohol-induced gastric lesions are due to stasis ingastric mucosa, which contributes to the development of thehemorrhage and necrotic aspects of the tissue injury (Shah et al.,2003; Raffin et al., 2007). The in vivo anti-ulcer evaluationdemonstrated that the enteric coated nanocapsules were able toreduce ulcer formation caused by oral administration of absoluteethanol (Fig. 5). The gastric mucosal protection against absoluteethanol can be mediated through a number of mechanisms thatinclude enhancement of the gastric mucosal defense throughincrease in mucus and/or bicarbonate production, reducing thevolume of gastric acid secretion or by simply neutralizing thegastric acidity (Senthilkumar et al., 2011).

434435436437438439440441442443444445446447448

Table 5Anti-ulcer activity of omeprazole enteric nanocapsules formula E3 compared toomeprazole suspension and control.

Group Ulcer index(Mean � SE)

Ulcer protection(%)

Control 5.69 � 1.14 –

Treatment 1 4.24 � 0.95 25.61Treatment 2 0.79 � 0.39 86.10


5. Conclusions

Enteric nanocapsules were successfully prepared by a novelnanoencapsulation technique. In contrast to classical coatingtechniques, the acid-labile drug, omeprazole could be successfullyencapsulated instantaneously in the enteric nanocapsules in astable state. The least particle size and polydispersity index values,highest zeta potential and encapsulation efficiency values wereobtained for formula E3 (containing 200 mg HPMCP). The in vivoevaluation corroborated with the in vitro results demonstratingthat omeprazole-loaded enteric nanocapsules were efficient inprotecting the stomach against ulcer formation. Further accelerat-ed stability study and comparative bioequivalence study in humanvolunteers against innovator will be conducted in the future toconfirm the formulation’s physical and chemical stability and itstherapeutic efficacy in humans.

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