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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

Nanoemulsion-based electrolyte triggered in situ gel for ocular delivery ofacetazolamide

Nadia Morsia, Magdy Ibrahima, Hanan Refaia,b,⁎, Heba El Sorogyb

a Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egyptb Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Misr University for Science and Technology, 6th October City, Egypt.

A R T I C L E I N F O

Keywords:AcetazolamideGellan gumGlaucomaIntraocular pressureIon sensitive in situ gelNanoemulsion

A B S T R A C T

In the present work the antiglaucoma drug, acetazolamide, was formulated as an ion induced nanoemulsion-based in situ gel for ocular delivery aiming a sustained drug release and an improved therapeutic efficacy.Different acetazolamide loaded nanoemulsion formulations were prepared using peanut oil, tween 80 and/orcremophor EL as surfactant in addition to transcutol P or propylene glycol as cosurfactant. Based onphysicochemical characterization, the nanoemulsion formulation containing mixed surfactants and transcutolP was selected to be incorporated into ion induced in situ gelling systems composed of gellan gum alone and incombination with xanthan gum, HPMC or carbopol. The nanoemulsion based in situ gels showed a significantlysustained drug release in comparison to the nanoemulsion. Gellan/xanthan and gellan/HPMC possessed goodstability at all studied temperatures, but gellan/carbopol showed partial drug precipitation upon storage and wastherefore excluded from the study. Gellan/xanthan and gellan/HPMC showed higher therapeutic efficacy andmore prolonged intraocular pressure lowering effect relative to that of commercial eye drops and oral tablet.Gellan/xanthan showed superiority over gellan/HPMC in all studied parameters and is thus considered as apromising mucoadhesive nanoemulsion-based ion induced in situ gelling formula for topical administration ofacetazolamide.

1. Introduction

Glaucoma is one of the leading causes of irreversible blindness inthe world. It is characterized by an increase in intraocular pressure(IOP) causing changes in optic nerve head and retinal nerve fiber layer(Sharma et al., 2008). Acetazolamide (AZA), a carbonic anhydraseinhibitor (CAIs), is used orally to treat glaucoma, by reducing theelevated IOP. AZA shows higher reduction (about 30%) in IOP whencompared to other CAIs e.g. methazolamide, brinzolamide and dorzo-lamide, which reduce IOP by about 20.3%, 21.5% and 23%, respec-tively (Singh et al., 2014). However, to obtain the desired lowering inIOP, large oral doses of AZA are used, which would cause peripheralinhibition of carbonic anhydrase enzyme that is distributed in almost allbody organs. This usually results in a wide array of side effects, whichare not tolerated by most of the patients and hence they discontinue thetherapy (Epstein and Grant, 1977). The most common reported sideeffects are diuresis, gastrointestinal symptoms including cramping,epigastric burning, nausea, diarrhea and metabolic acidosis (Graneroet al., 2008). The poor aqueous solubility and low corneal permeabilityof the drug limits its ocular bioavailability, causing an insufficient

amount of the drug to reach the ciliary body. Many researchers adapteda number of approaches to enhance its ocular bioavailability in order todevelop an effective topical AZA formulation. These attempts includeformulations of AZA in the form of aqueous solutions containingcyclodextrins or penetration enhancers, polymeric suspension andvesicular preparations as niosomal and liposomal dispersions(Loftsson et al., 1994; Kaur and Smitha, 2002; Guinedi et al., 2005;Hathout et al., 2007).

Nanoemulsions (NEs), particularly, oil/water nanoemulsions havebeen investigated as a potential drug delivery system in ophthalmology(Ghosh and Murthy, 2006). Earlier successful nanoemulsions for oculardelivery of pilocarpine (Ince et al., 2015), timolol maleate (Gallarateet al., 2013) and dorzolamide (Ammar et al., 2009) have beenformulated. NEs possess unique physicochemical properties namely,high solubilizing capacity for various drugs as well as acting aspenetration enhancers to facilitate corneal drug delivery. Furthermore,the low surface tension of NEs would also guarantee a good spreadingeffect on the cornea and a proper mixing with the precorneal filmconstituents, thus would possibly improve the contact between the drugand corneal epithelium. These properties make NEs an ideal vehicle for

http://dx.doi.org/10.1016/j.ejps.2017.04.013Received 2 February 2017; Received in revised form 24 March 2017; Accepted 18 April 2017

⁎ Corresponding author at: Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Misr University for Science and Technology, 6th of October City, Egypt.E-mail address: hanan.refai@must.edu.eg (H. Refai).

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Available online 19 April 20170928-0987/ © 2017 Published by Elsevier B.V.

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the topical delivery of AZA, which possess very poor topical effective-ness due to its very slight solubility in aqueous tear fluid (0.7 mg/ml)and its very low corneal penetration coefficient (4.1 × 10−6 cm/s)(Granero et al., 2008).

However, one of the main problems encountered with the topicaldelivery of ophthalmic drugs is the rapid and extensive precorneal losscaused by the drainage and the high tear fluid turnover. As aconsequence, the ocular residence time of conventional eye drops islimited to few minutes (Kumar et al., 1994) and the ocular absorptionof a topically applied drug is reduced to approximately 5% (Gaudanaet al., 2009). The drained drug is mainly absorbed systemically viaconjunctiva and nasal mucosa, which may result in the undesiredperipheral side effects of the drug (Kumar et al., 1994). To overcomethese problems an increase in the contact time between dosage formand corneal surface is required.

In situ gelling systems are viscous liquids, which undergo a sol–gelphase transition, when applied to human body, due to change in aphysicochemical parameter such as temperature, pH or ionic strength(Robinson and Mlynek, 1995). They allow accurate and reproducibleadministration of a drug unlike preformed gels, and are capable ofprolonging the formulation's residence time to the mucosal surface dueto post-administration gelling (Krauland et al., 2003).

In our previous study (Morsi et al., 2014) AZA loaded NE systemscomposed of either oleic acid, isopropyl myristate or peanut oil at 40%water content were formulated. The in vitro release and ex vivopermeation studies showed superiority of peanut oil over other oilsystems. The optimized formula was also formulated at higher watercontent which revealed lower ocular irritation but reduced ocularretention.

The aim of the present work was to thoroughly characterize AZA NEof peanut oil at different surfactant and cosurfactant combinations athigher water content and develop a novel ophthalmic NE based in situgelling system to exploit the benefits of NE in enhancing drugsolubilization and permeation across corneal membrane in addition toprolonging the contact time of the formulation to the corneal surface.The work mainly focused on ion-activated NE based in situ gellingsystems using gellan gum polymer alone and in combination with otherpolymers.

2. Materials and methods

2.1. Materials

Acetazolamide (99.9% purity) was obtained from CID Company,Egypt. Tween 80 was obtained from Gomhorya Company, Egypt.Peanut oil (refined arachis oil) was purchased from Nefertari BodyCare, Egypt. Transcutol P (diethylene glycol monoethyl ether) waskindly donated by Gattefossé, France. Cremophor EL (polyoxyl 35castor oil) was kindly donated by BASF, Germany. Gellan gum andphosphotungstic acid hydrate were procured from Fluka Bio Chemika,Switzerland. Xanthan gum, potassium dihydrogen phosphate, disodiumhydrogen phosphate, sodium bicarbonate, calcium chloride and sodiumchloride were purchased from El-Nasr Pharmaceutical Chemicals Co.,Egypt. Carbopol 940 and hydroxypropyl methylcellulose (HPMC) weredonated by Colorcon, UK.

2.2. Methods

2.2.1. Formulation of AZA loaded nanoemulsions2.2.1.1. Construction of pseudoternary phase diagram. Six pseudoternarysystems consisting of oil (peanut oil), surfactant (tween 80, cremophorEL or their mixture at 1:1 weight ratio), and cosurfactant (propyleneglycol or transcutol P) were prepared using water titration method at25 °C (Garti et al., 2000). The corresponding pseudoternary phasediagrams were constructed using Grapher Golden Software (Version8.1.388, US). The ratio of surfactant to cosurfactant (S:CoS) was fixed at

9:1. Blends of oil and S:CoS mixture were prepared at different ratiosstarting from 0.5:9.5 to 9:1. Samples were left for equilibrium after eachaddition of water then they were visually observed for clarity andflowability (Tayel et al., 2013). The area representing the clear NEregion in each phase diagram was calculated using AutoCAD® software(Autodesk Inc., San Rafael, US).

2.2.1.2. Preparation of AZA loaded NEs. Based on pseudoternary phasediagrams, NE formulae were selected to be loaded with AZA from eachof the six studied systems at (S/CoS):oil ratio 9.5:0.5 and at 60% watercontent. In order to simulate physiological dilution process by tearsafter ocular administration, NEs were subjected to dilution withsimulated tear fluid (STF) in a ratio of 1:5 (v/v) and assessed visuallyfor transparency for a period of 48 h (Ammar et al., 2010). The STF(based on electrolyte composition of tears) was prepared by dissolving6.8 g NaCl, 2.2 g NaHCO3, 0.084 g CaCl2·2H2O and 1.4 g KCl in 1 l ofdeionized water. These amounts are equal to 142 mM of Na+, 19 mM ofK+ and 0.6 mM of Ca2+ with an osmolality of 288 ± 5 mmol/kg (Qiet al., 2007).

To prepare drug loaded NE, 1% w/w AZA was sonicated withsurfactant/cosurfactant/oil blends for 30 min until complete dissolu-tion of the drug then the aqueous phase was added drop wise. Thedeveloped NE formulae were also subjected to examination under crosspolarized microscope (Olympus, Japan) to exclude liquid crystallinesystems and to examine and verify the isotropic behavior of NEs. Thecompositions of different in NE systems are shown in Table 1.

2.2.2. Development of AZA loaded NE based in situ gel2.2.2.1. Formulation of in situ gelling systems using only gellan gum. Theselected AZA loaded NE was prepared with only 5% of its water contentusing deionized water. Different concentrations of gellan gum weredissolved by heating at 90 °C for 20 min in the remaining amount ofwater of the NE then allowed to cool at room temperature. The in situgelling systems were prepared by dispersing NE into the gel solution toobtain final gellan gum concentrations of 0.1, 0.2, 0.3, 0.4, 0.5 and0.6%.

The in situ gelling systems were evaluated for their gelling capacityto identify the most suitable composition. In order to mimic thesituation occurring upon ocular instillation, the formulations werediluted with freshly prepared STF in the proportion of 25:7 (25 μl:volume of the formulation; 7 μl: normal volume of tear fluid in the eye)and the gelation was assessed by visual inspection and graded asfollows(Carlfors et al., 1998):

(−) no gelation(+) slow weak gelation(++) immediate gelation that lasts for 2–3 h(++

+)immediate stiff gelation which remains for extended period(6–8 h) of time

Table 1Composition of AZA loaded NE bases.

NE base Composition % (w/w)

AZA Peanut oil Tw 80 Cr EL Trans PG W

NE1 1 2 34.2 – – 3.8 59NE2 1 2 34.2 – 3.8 – 59NE3 1 2 – 34.2 – 3.8 59NE4 1 2 – 34.2 3.8 – 59NE5 1 2 17.1 17.1 – 3.8 59NE6 1 2 17.1 17.1 3.8 – 59

NE: nanoemulsion, Tw 80: tween 80, Cr EL: cremophor EL, Trans: transcutol P, PG:propylene glycol, W: deionized water.

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2.2.2.2. Formulation of in situ gelling systems using combined polymers. Insitu gelling systems were prepared using three different polymers(xanthan gum, HPMC K4 M and carbopol 940) in combination withgellan gum.

In the case of gellan/xanthan and gellan/HPMC systems, thepolymers were dissolved in deionized water by heating at 90 °C for20 min then allowed to cool at room temperature. For the preparationof the systems containing carbopol 940, the polymer was sprinkled andallowed to hydrate in cold deionized water and then mixed with gellangum solution. The AZA loaded NE was then dispersed into the polymersolution to obtain a final polymer concentration of 0.2, 0.4 or 0.6% w/w (xanthan, HPMC or carbopol) together with 0.3% gellan gum. Thecompositions of different in situ gelling systems are shown in Table 2.

2.2.3. Physicochemical characterization of the developed formulae2.2.3.1. Droplet size, polydispersity index and zeta potential. The dropletsize, size distribution and zeta potential were determined by a zetasizer(Zetasizer Nano ZS, Ver. 6.20, Malvern Instrument Ltd., UK). Allmeasurements were performed in triplicate at room temperature(25 °C) after diluting the samples by 10-folds.

2.2.3.2. pH measurement. Measurements of pH were done by pH meter(JENWAY model 350, JENWAY Ltd., UK).

2.2.3.3. Refractive index. Refractive index was determined at 25 °Cusing high precision digital automatic refractometer (RFM870,Bellingham and Stanley Ltd., UK).

2.2.3.4. Surface tension. Surface tension measurement was carried outat 25 °C using a thermostatically controlled processor tensiometer K100(Kruss GmbH, Germany).

2.2.3.5. In vitro release. The in vitro release studies were performed intriplicate using a USP dissolution tester apparatus (Hanson RS8-plus,US) adjusted at 34 ± 0.5 °C to simulate the ocular surfacetemperature. Half gram of each formula loaded with AZA (1% w/w)was placed in a glass cylindrical tube (2.5 cm in diameter and 10 cm inlength) tightly covered with a semi-permeable membrane (SpectraPor©, Spectrum Lab, US) from one end. The loaded tubes wereattached to the shafts of the USP dissolution tester from the other endso that the membrane covered ends of the tubes were just immersed inthe reservoir. The shafts were rotated at a speed of 50 rpm in 100 mlsimulated tear fluid (pH 7.4). Periodically, 1 ml samples werewithdrawn at predetermined time intervals of 0.5, 1, 2, 3, 4, 5, 6, 7and 8 h. Replacement with fresh medium was ensured to maintain aconstant volume. The samples were analyzed spectrophotometrically(UV spectrophotometer; Shimadzu, USA) at 263 nm and the percentdrug released was plotted versus time.

AZA release profiles were fitted to zero order, first order and

Higuchi diffusion models to estimate the best fitting kinetic modelhaving the highest correlation coefficient.

2.2.3.6. Evaluation of rheological properties. Rheological characteristicsof the prepared formulae were determined using a cone and plateviscometer (Brookfield DV III, US). For liquid formulae the viscometerwas fitted with a cone spindle 40 and each sample was investigated atshear rates from 5 to 50 rpm (keeping a period of 10 s at each rpm). Inthe case of gelled systems the viscometer was fitted with spindle 52 andeach sample was investigated at shear rates from10 to 100 rpm. Allmeasurements were performed in triplicates.

2.2.3.7. Drug content uniformity. Accurate weight of each formulation(n = 3) was diluted with DMSO/water (1:1) and the absorbance wasmeasured at 267 nm by using UV visible spectrophotometer (Shimadzu,USA).

2.2.3.8. Degree of transparency. The prepared AZA loaded NE in situgelling formulae were inspected for optical transparency by measuringpercentage transmittance (%T) spectrophotometrically (UVspectrophotometer; Shimadzu, USA) at λ = 520 nm using thefollowing equation (Tayel et al., 2013):

A = 2 − log(%T)

where A is the measured absorbance of the formulae against a blank(deionized water).

2.2.3.9. Mucoadhesive strength. A modified balance method (Koffi et al.,2006) was used to determine the mucoadhesive performance ofdifferent formulations by measuring the force required to detach thegel from a mucosal surface. The instrument was composed of a modifiedtwo arms balance in which the right pan had been attached to a glassvial. Rabbit intestinal mucosa was used as a model mucous membrane(Refai and Tag, 2011) for bioadhesion testing. It was dissected, washedthen placed in 0.9% NaCl solution. Mucosal membranes were instantlysecured with the mucosal side out onto the glass vial using a rubberband. This was followed by tarring the balance. Half gram of the gelwas spread on an area of 1 cm2 on another piece of mucosa, which wasadhered to another glass vial put on a moving platform. The platformwas slowly raised until the gel touched the upper mucosa. The gel andmucosa were left in contact for 2 min to allow the formation of adhesivebonds, after which weights were added to the left pan. Addition ofweights was stopped upon detachment of the gel from the mucosa. Theweight causing the detachment was recorded. The mucoadhesivestrength was measured according to the following equation (Mahajanand Gattani, 2010):

⎜ ⎟⎛⎝

⎞⎠Mucoadhesive strength dyne

cm= m g

A2

∗

Table 2Composition of different in situ gelling formulae.

In situ gel formula Composition % (w/w)

NEa Gellan gum Xanthan gum HPMC K4M Carbopol 940 Deionized water

F1 46 0.3 – – – 53.7F2 46 0.3 0.2 – – 53.5F3 46 0.3 0.4 – – 53.3F4 46 0.3 0.6 – – 53.1F5 46 0.3 – 0.2 – 53.5F6 46 0.3 – 0.4 – 53.3F7 46 0.3 – 0.6 – 53.1F8 46 0.3 – – 0.2 53.5F9 46 0.3 – – 0.4 53.3F10 46 0.3 – – 0.6 53.1

a NE composition is 17.1% tween 80, 17.1% cremophor EL, 3.8% transcutol P, 2% peanut oil, 1% AZA, 5% deionized water.

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where m is the weight required for detachment in grams, g is theacceleration due to gravity (980 cm/s2) and A is the surface area ofmucosa exposed to the formulation (cm2). The experiment wasperformed in triplicate.

2.2.3.10. Gel strength determination. A method described by (Mahajanand Gattani, 2010) was used to determine the gel strength of theformulated NE based in situ gels. A sample of 50 g was placed in a 100-ml graduated measuring cylinder (d = 3.75) and gelled in athermostatically controlled water bath at 32–34 °C by addition ofSTF. A weight of 27 g was placed onto a disk (d = 3.5 cm;thickness = 0.3 cm) and this disc was put onto the gel surface. Thegel strength was measured as the time (seconds) required for the disc tosink 5 cm down through the gel.

2.2.3.11. Transmission electron microscopy (TEM). Surface morphologyof NE as well as NE based in situ gels was studied by transmissionelectron microscopy (JOEL JEM-1230, Japan) operating at 200 kVcapable of point-to-point resolution. One drop of each drug loadedformula was loaded onto a copper grid and the excess was removedwith a filter paper. The formulae were stained with one drop ofphosphotungstic acid aqueous solution (2% w/v) for 3 min priorexamination.

2.2.3.12. Thermodynamic stability studies. For stability studies, the NEbased in situ gelling formulations were prepared as mentioned beforeand 0.02% w/w methyl paraben was added as a preservative. Theformulations were sterilized by autoclaving (Remi Equipments Pvt.Ltd., Mumbai, India) at 121 °C for 20 min at 15 psi. Stability studieswere carried out at 40, 25 and 4 °C for 3 months. Samples werewithdrawn every 1 month and evaluated for appearance, viscosity,drug content and pH. The value of each observation was compared withfreshly prepared samples.

2.2.3.13. In vivo studies. Ocular irritation and pharmacodynamicstudies of AZA loaded NE based in situ gelling formulations weredone on adult male New Zealand albino rabbits. The protocol of thestudies was approved by the Research Ethics Committee, Faculty ofPharmacy, Cairo University, Egypt and they comply with the ARRIVEguidelines. The rabbits weighing about 2–3 kg were housedindividually at the standard environmental conditions (dark:lightcycle 12 h each, 20–25 °C temperature and 40–70% relativehumidity). Rabbits were fed with standard pellet diet and water wasprovided ad libitum. All animals used in the experiment were healthyand free from any clinically observable ocular abnormalities.

2.2.3.13.1. Ocular irritation testing. The test was conductedaccording to the modified Draize test (Baeyens et al., 2002). All theglass wares used in the experiment and all applied formulations weresterilized by autoclaving. Nine New Zealand albino rabbits weredivided into three groups of 3 rabbits each. The first group receivednormal saline and served as control. The second and third groupsreceived gellan/xanthan (F2) and gellan/HPMC (F5), respectively. Onedrop (25 μl) of the NE based in situ gelling formulation was instilledinto the lower cul-de-sac of one eye of each rabbit. The untreatedcontra-lateral eye was used as a control. The eyelids were gently heldtogether for about 10 s to avoid the loss of instilled preparations. Eachanimal was observed for ocular reactions (redness, discharge,conjunctival chemosis, iris and corneal lesions) at 5, 10, 15, 30 minand 1, 2, 3, 6, 9, 12, 24 h post instillation. The following scores wereused to evaluate the irritation (Shell, 1982):

0: No redness, no inflammation or excessive tearing1: Mild redness with inflammation and slight tearing2: Moderate redness with moderate inflammation and excessive

tearing

3: Severe redness with severe inflammation and excessive tearing

The overall ocular irritation index (Iirr) was calculated by summingup the total clinical evaluation scores over the observation time points.A score of 2 or 3 in any category or (Iirr)> 4 was considered as anindicator of clinically significant irritation.

2.2.3.13.2. In vivo pharmacodynamic studies (therapeutic efficacystudies). Twelve New Zealand albino rabbits were randomized in fourgroups having three in each. Glaucoma was induced to the rabbits by anintraocular injection of 0.25 ml of 2% w/w sodium carboxymethylcellulose (NaCMC) prepared by dissolving NaCMC in pyrogenfree water for injection (Zhu and Cai, 1992). The intraocular pressure(IOP) of the rabbits was measured using a tonometer (Schoetztonometer, Teufel, Germany). Before taking the measurement, eyeswere anesthetized with 1–2 drops of benoxinate hydrochloride(Benox®) eye drops. After intraocular injection of NaCMC, the eyeswere left for 48 h to develop glaucoma then the IOP was measured atvarious times (1, 2, 6, 10 and 24 h) to establish the IOP baseline of eachglaucomatous rabbit's eye before the application of treatment. A singledose of 50 μl of the selected formulae loaded with 1% AZA was appliedtopically to the cornea of the left eye of the first two groups. Forcomparison, 50 μl of Azopt® (brinzolamide, 1% w/v) were administeredto the left eyes of rabbits of the third group. The right eyes of the firstthree groups received no medication and served as control. The fourthgroup received orally a fraction of AZA tablet (Cidamex®) equivalent to9 mg AZA (calculated dose for rabbits of average weight of 2.5 kg). TheIOP was measured at 0.5, 1, 1.5, 2, 3 4, 5, 6, 7, 8, 9 and 10 h afterreceiving the medication. The percentage decrease in IOP for each timepoint was calculated as follows:

%IOP decrease = IOP before treatment − IOP after treatmentIOP before treatment

× 100

The greatest reduction in % IOP was termed as the “maximum %decrease in IOP”. The therapeutic profiles (% reduction of IOP versustime plots) of AZA in situ gels versus AZA tablet and brinzolamide eyedrops were used to calculate the pharmacodynamic parameters: areaunder the curve (AUC0–10), time required to achieve peak IOP reduction(tmax) using WinNonlin® software (Ver. 1.5, Scientific consulting Inc.,Cary, NC, US).

3. Results and discussion

3.1. Development of AZA loaded nanoemulsions

Six pseudoternary systems consisting of oil (peanut oil), surfactant(tween 80, cremophor EL or their mixture at 1:1 weight ratio), andcosurfactant (propylene glycol or transcutol P) were prepared at anoptimum S/CoS ratio of 9:1 (Morsi et al., 2014). According topseudoternary phase diagrams, NE formulae were selected from eachof the six studied systems at (S/CoS):oil ratio of 9.5:0.5 and 60% watercontent in order to maintain NE physical integrity upon dilution withsimulated tear fluid in the ratio of 1:5 (v/v) (Fig. 1). The prepared NEswere loaded with 1% (w/w) AZA. In all prepared NEs, no liquidcrystalline domains were observed under polarized microscope. TheAZA loaded NEs were characterized to select the optimum NE formulato be incorporated in in situ gelling system.

3.2. Characterization of nanoemulsion formulations

3.2.1. Droplet size, polydispersity index and zeta potentialThe NE formulations showed a relatively small droplet size ranging

from about 11 to 15 nm. From the results it is noticed that, the type ofsurfactant had a slight influence on droplet size, while the cosurfactantsrole was insignificant (Table 3). NE1 and NE2 containing tween 80showed smaller droplet sizes than NE3 and NE4 that are prepared withcremophor EL, while the mixture of both surfactants (NE5 and NE6)

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resulted in the formation of droplets of intermediate size (p < 0.05).This result may be due to the difference in HLB value of the surfactants.The HLB value for tween 80 and cremophor EL are 15 and 13,respectively (Buyukozturk et al., 2010). Weerapol et al. (2014)observed that the emulsion droplet size decreased with a higher HLB.Higher HLB values indicate that surfactants have higher hydrophilicity,

which facilitates reduction in the curvature of the interface for the oilleading to smaller droplet size (Kong et al., 2011). The small PDI valuesobserved for all the formulations (Table 3) indicate that the NE dropletswere homogenous and had narrow size distribution. Despite thenonionic nature of the surfactants and the deionized quality of water,the dispersed droplets acquired a negative charge (Table 3). This charge

Fig. 1. Pseudo-ternary phase diagrams of nanoemulsion composed of a) peanut oil/tween 80/transcutol P, b) peanut oil/tween 80/propylene glycol c) peanut oil/cremophor EL/transcutol P, d) peanut oil/cremophor EL/propylene glycol e) peanut oil/tween 80-cremophor EL/transcutol P, f) peanut oil/tween 80-cremophor EL/propylene glycol. O: peanut oil, W:water, S/CoS: surfactant/cosurfactant mixture at 9:1 ratio, NE: nanoemulsion existence area, NEG: nanoemulsion gel existence area.

Table 3Droplet characteristics, viscosity and refractive index of AZA loaded NEs (mean ± SD, n = 3).

NE formula Droplet diameter (nm) Zeta potential (mV) Polydispersity index (PDI) Viscosity (cp) Refractive index

NE1 10.97 ± 0.23 −10.89 ± 2.28 0.26 ± 0.035 36.7 ± 0.3 1.3892 ± 0.0001NE2 11.18 ± 0.49 −10.81 ± 1.97 0.27 ± 0.035 64.1 ± 1.5 1.3886 ± 0.0002NE3 15.42 ± 0.21 −1.49 ± 0.30 0.23 ± 0.014 68.2 ± 2.4 1.3889 ± 0.0002NE4 15.14 ± 0.18 −2.33 ± 0.07 0.39 ± 0.028 87.5 ± 5.6 1.3890 ± 0.0003NE5 13.58 ± 0.08 −6.69 ± 1.40 0.25 ± 0.034 68.3 ± 4.1 1.3889 ± 0.0001NE6 13.46 ± 0.12 −4.49 ± 1.48 0.26 ± 0.035 83.8 ± 3.3 1.3889 ± 0.0002

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may be due to the presence of surface active ionic contaminants, whichcame along with the nonionic surfactants. It is also believed that, whennonionic surfactants are used for emulsification, the OH− ions,produced by the dissociation of water molecules, are preferentiallyadsorbed at the surface of dispersed droplets. This is influenced byhydrocarbon chain length and/or the polar group of the nonionicsurfactant molecule (Manev and Pugh, 1991).

3.2.2. Refractive indexThe refractive index of eye drops should ideally be close to that of

the tear fluid (1.34–1.36) to ensure a clear vision and good patientcompliance (Fialho and da Silva-Cunha, 2004). However, Keipert et al.(1989) reported that, the refractive index of eye drops could exceed thisrange to a maximum of 1.47 without a noticeable discomfort to thepatient. From Table 3 it is to be noticed that the refractive indices of allAZA loaded NEs were within the acceptable range.

3.2.3. ViscosityThe viscosity of AZA loaded NE formulae was measured at mini-

mum shear rate of 37.5 s−1. The systems revealed in general lowviscosity values due to their consistence of high proportion of water(Table 3). NEs prepared using cremophor EL showed higher viscositiesthan those prepared using tween 80. This is probably due to higherviscosity of cremophor EL (~700 mPa·s) in contrast to tween 80(~400 mPa·s). Also, the nature of cosurfactant seemed to influencethe viscosity of the systems. Using PG obviously lowered the viscosity ofthe NEs which is probably attributed to its ability to form hydrogenbonding with surfactant head groups, which reduces the ability of thehydroxyl groups of PG to form hydrogen bonds with water. Accord-ingly, the presence of the PG molecules located at oil-water interface ofthe NE decreases the fluidity of the interfacial film hence reducing theapparent viscosity of the system (Resende et al., 2008). With respect toflow behavior, all NE systems showed pseudoplastic flow whichindicates progressive rupture of the internal structure of the preparation(by increasing shear) and its later reconstruction by means of Brownianmovement.

3.2.4. pHThe ideal pH for an ophthalmic preparation when instilled in the

eye should be in the range of 7.2 ± 0.2 (Ammar et al., 2009).However, pH values from 3.5 to 8.5 can be tolerated if the preparationis not or is only very slightly buffered because in this case the limitedbuffering capacity of the tears is able to adjust the pH to physiologiclevels on administration (Ammar et al., 2009). The pH values of theprepared AZA NE were in the range of 5.4 to 5.7 (Table 4) beingtherefore adequate for their application to the eye because the preparedNEs are not buffered and could be adjusted to the physiological valuesby tears. The obtained values were also able to maintain drug stability,as AZA is highly unstable at alkaline pH values and has maximumstability at pH values 4–5 (Khamis et al., 1993).

3.2.5. Surface tensionThe physiological value of the lacrimal fluid surface tension ranges

from 42 to 46 mN/min (Fialho and da Silva-Cunha, 2004). Theadministration of eye drops with lower surface tension than that of

the lacrimal fluid results in destabilization of the tear film (Lin andBrenner, 1982), which would guarantee a good spreading effect on thecornea and good mixing with the precorneal film constituents, thuspossibly improving the contact between the drug and the cornealepithelium (Haβe and Keipert, 1997). From the results listed in Table 4it is obvious that the NEs containing tween 80 alone or in combinationwith cremophor EL are more preferred for ocular administration thanthose containing only cremophor as they showed lower surface tensionvalues than those of the tear fluid (p < 0.05). This indicates thegreater surface active property of tween 80 over cremophor probablydue to higher HLB value. This finding is consistent with the smallerdroplet sizes observed for the corresponding NEs (Table 3).

3.2.6. In vitro releaseTo facilitate comparison between release behaviors of different

formulations, percent of drug released at 1.5 h, mean dissolution rate(MDR) and the area under percent drug released time curve (AUC) werecalculated and the results are represented in Table 4. Results show that,the in vitro release patterns of all formulae were similar. This isprobably due to the very close droplet sizes of the NEs. The releasereached about 80% after 3 h, which is attributed to the very smalldroplet size range of the NEs, resulting in an increase of total number ofoil globules and subsequent increase in their surface area. However,NE6 which is composed of surfactant mixture and transcutol P ascosurfactant showed a significantly higher drug release (p < 0.05)compared to other NEs. This combination of surfactants and cosurfac-tant must have increased the fluidity of the interface permitting higherdrug release (Agubata et al., 2014).

In order to study the effect of nanoemulsion based electrolytetriggered in situ gel as drug delivery system for AZA, NE6 was selectedfor the incorporation of in situ gelling polymers. The different NEformulae did not show valuable differences in their characteristics, butNE6 containing tween 80 and cremophor RL as a surfactant mixture andtranscutol P as cosurfactant seemed most appropriate for furtherinvestigations based on the high NE existence area, low surface tensionand high drug release revealed by this formula.

3.3. Development of AZA loaded NE based in situ gel

3.3.1. Gelation capacity of NE based gellan gum in situ gelsAn ideal in situ gelling ocular delivery system should be a free

flowing liquid with low viscosity at non-physiological condition toallow reproducible administration into the eye as drops but undergo insitu phase transition to form gel capable of withstanding shear forces inthe cul de-sac and sustain drug release at physiological condition(Kumar and Himmelstein, 1995). Gelation capacity of NE based gellangum in situ gels was evaluated using different concentrations of gellangum in order to select the concentration of gellan gum that givesoptimum gelation i.e. immediate stiff gelation which remains for anextended period of time. As shown in Table 5, the concentration of0.1% gellan gum took the score (−), which indicates the inability of thepolymer at this concentration to produce gelation upon dilution withSTF. Also, 0.2% of the polymer was insufficient to produce gel withpromising gelation characteristics. An increase in polymer concentra-tion to 0.3 and 0.4% produced gels with suitable strengths when diluted

Table 4In vitro release parameters, pH and surface tension of AZA loaded NEs (mean ± SD, n = 3).

NE formula AUC (μg/ml·h) Release at 1.5 h (%) MDR (μg/ml/h) pH Surface tension (mN/m)

NE1 182.28 ± 4.82 70.71 ± 2.78 1.04 ± 0.01 5.7 ± 0.0 34.52 ± 0.46NE2 184.87 ± 3.66 67.63 ± 2.77 1.07 ± 0.01 5.5 ± 0.1 34.36 ± 0.21NE3 178.18 ± 3.08 70.56 ± 4.45 0.98 ± 0.03 5.4 ± 0.0 42.39 ± 0.06NE4 184.02 ± 2.97 71.74 ± 2.45 1.06 ± 0.02 5.4 ± 0.2 41.50 ± 1.72NE5 187.52 ± 3.69 69.27 ± 2.87 1.07 ± 0.02 5.6 ± 0.0 38.77 ± 0.75NE6 199.07 ± 2.90 75.18 ± 1.63 1.18 ± 0.05 5.5 ± 0.0 37.92 ± 0.53

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with STF and they were free flowing liquids before dilution. A furtherincrease in gellan concentration to 0.5 and 0.6% formed stiff gel even atnon physiological condition. The minimum possible concentration ofgellan gum, which produced gel with the required properties (0.3%)was selected as the optimum in situ gelling concentration and was usedto study the effect of other polymers on the physicochemical propertiesof the in situ formed gels.

3.3.2. Formulation of in situ gelling systems using mixed polymersThe optimized concentration of gellan gum (0.3%) was used to

prepare NE based in situ gelling formulae in combination with threedifferent mucoadhesive polymers (xanthan gum, HPMC K4 M andcarbopol 940), that are known to exhibit in situ gelation by differentmechanisms, in order to study the possible synergistic effect caused bythe polymer combinations and to improve the mucoadhesive propertiesof the in situ gelling formula.

3.4. Physicochemical characterization of NE based in situ gelling systems

3.4.1. Drug content uniformityThe percent drug content for all formulations was found to be

satisfactory in the range of 97–102% (data not shown).

3.4.2. pHThe pH of NE based in situ gels was found to be between 4.7 and 5.8

(Table 6), being therefore adequate to their application to the eye(Shanmugam et al., 2011). The obtained values were also able topreserve stability of AZA (Khamis et al., 1993). It is also obvious that,the combination of either xanthan gum or HPMC to gellan in situ gelhad no significant effect on the pH, whereas a successive reduction ofpH was observed with increased carbopol concentration, which isattributed to the acidic nature of the polymer.

3.4.3. Rheological behaviorViscosity and gelling capacity are the most important factors

evaluating the successfulness of in situ gelling systems. For an easyinstillation in the eye, the formulation must have optimum viscosityand for a prolonged residence time, it should undergo rapid sol to geltransition upon contact with tear fluid. Since, the ocular shear rate isvery large, ranging from 0.03 s−1 during interblinking periods to4250–28,500 s−1 during blinking (Srividya et al., 2001), the formula-tions with pseudoplastic rheological characteristics are usually pre-ferred for ocular delivery. The rheological behavior of the in situ gellingformulae was evaluated and it was found that all formulations exhibiteda shear thinning pseudoplastic behavior in both non-physiologic (beforegelation) and physiologic (after gelation) conditions. It is thereforeexpected that such formulations will not present difficulty in blinkingwhile undergoing gelation in the eye.

3.4.3.1. Viscosity of the in situ gelling systems in the sol state. Bycomparing rheograms of different formulations in the sol state, it wasfound that the addition of in situ gelling polymers to the NE caused anobvious increase in viscosity (p < 0.001). The extent of viscosityincrease was related to both, type and concentration of polymers(Table 6). As the concentration of any polymer was increased, theviscosity of formulation was also increased. The lowest viscosity valuewas observed for (F1), which contained gellan gum polymer alone.With respect to the type of combined polymer, it was found that theincrease in viscosity could be ranked in the order ofxanthan > carbopol > HPMC. The remarkable high viscosity ofxanthan may be attributed to its anionic nature. The electrostaticrepulsions from the charged groups on the side chains make itsmolecules extend (Khouryieh et al., 2007). Because of this, themolecules align and associate via hydrogen bonding to form a weaklystructured helical conformation which would immobilize the free waterand increase the viscosity. In addition, the higher viscosity of xanthangum is probably also related to its high molecular weight(approximately two million Daltons) (Dintzis et al., 1970) whichincreases the intermolecular association among polymer chains.Although, carbopol is also anionic in nature, the majority of thecarboxylic groups are not dissociated at pH values below the pKa ofthe polymer which is about 5.5, thus the molecules are not sufficientlyextended, which results in reduced swelling and low viscosity (Kuteet al., 2015). HPMC is a neutral polymer and the viscosity of its solutionis the result of hydration of polymer chains through hydrogen bonding,causing them to extend and form relatively open random coils. TheHPMC grade employed in the present study was of Methocel K4M type,which is not a high molecular weight grade with a correspondingintermediate viscosity (Ford, 2014).

3.4.3.2. Viscosity of in situ gelling systems in the gel state. The addition ofSTF to all formulations resulted in an intense increase in viscosity dueto gelation. By comparing viscosity values of formulae containingcombined polymers (F2–F10) with that containing only gellan gum(F1), only a slight increase in viscosity could be detected. This leads to

Table 5In situ gelling capacity of NE based in situ gels prepared using different concentrations ofgellan gum.

Formulation code Gellan gumconcentration (%)

Gelling capacity

Before additionof STFa

After additionof STF

G1 0.1 (−) (−)G2 0.2 (−) (++)G3 0.3 (−) (+++)G4 0.4 (−) (+++)G5 0.5 (+++) (−)G6 0.6 (+++) (−)

a Simulated tear fluid.

Table 6Physicochemical properties of NE based in situ gels (mean ± SD, n = 3).

Formula pH Viscosity at min shear rate (cp) Gel strength (s) Mucoadhesive strength (dyne/cm2)

Sol Gel Sol Gel

F1 5.6 ± 0.2 6.2 ± 0.2 164.8 ± 12.2 6984.5 ± 23.7 33.5 ± 1.2 3012.7 ± 22.0F2 5.8 ± 0.0 6.3 ± 0.1 614.8 ± 7.9 7461.5 ± 35.4 35.1 ± 0.3 5817.7 ± 19.3F3 5.8 ± 0.1 6.3 ± 0.0 761.3 ± 10.1 7598.0 ± 11.9 38.2 ± 0.7 6752.9 ± 45.8F4 5.8 ± 0.0 6.4 ± 0.0 1234.5 ± 35.4 9175.5 ± 51.1 41.4 ± 2.1 8276.6 ± 28.4F5 5.8 ± 0.0 6.5 ± 0.1 177.9 ± 21.2 7105.0 ± 22.2 34.9 ± 0.9 3878.6 ± 54.7F6 5.7 ± 0.1 6.5 ± 0.0 347.9 ± 14.3 7519.5 ± 16.4 35.4 ± 0.5 4467.3 ± 47.1F7 5.8 ± 0.0 6.5 ± 0.0 528.5 ± 9.9 7715.0 ± 10.8 37.6 ± 1.1 4709.7 ± 23.6F8 5.2 ± 0.0 5.8 ± 0.2 410.8 ± 19.8 7975.0 ± 17.2 34.0 ± 2.2 6510.4 ± 11.5F9 4.9 ± 0.1 5.4 ± 0.0 507.5 ± 9.5 6470.5 ± 21.1 31.9 ± 0.8 6649.0 ± 14.1F10 4.7 ± 0.0 5.0 ± 0.1 643.6 ± 12.6 5346.0 ± 33.5 29.4 ± 0.6 4882.8 ± 18.2

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the conclusion that, in situ gelation of different formulations wasmainly due to the presence of gellan gum. Gellan gum forms doublehelices in an ion-free aqueous medium, which are loosely associatedwith each other by van-der-Waals attraction, giving the formulationlow viscosity. Once in contact with the cations of the tear fluid, some ofthe helices associate into cation-mediated aggregates, which cross-linkthe polymer resulting in gel formation (Nanjawade et al., 2007).Although xanthan gum is known as an ion induced in situ gellingpolymer, the applied concentrations seemed to be less influenced bycations than gellan gum. Higher concentrations of xanthan gum wereprobably needed to achieve gel formation. This result is in accordancewith that detected by Rupenthal et al. (2011), who reportedthat> 0.5% xanthan gum concentration is needed to achieve in situgelation. This may be explained by the branched nature of the xanthangum backbone, with the trisaccharide side chain preventing helixformation at low polymer concentrations. Nevertheless theconcentration of xanthan gum could not be increased beyond 0.6% asit would have caused gelling of the original formula.

HPMC as a thermo-sensitive polymer, caused only a slight increasein viscosity after mixing formulations (F5–F7) with STF at 35 °C. HPMCmolecules probably need higher temperatures to associate and to formhydrophobic interactions between methoxy substitutions (Gambhireet al., 2013).

Carbopol is a well-known pH induced in situ gelling polymer.Although, its combination with formulations with gellan gum showedthe highest increase in viscosity at 0.2% polymer concentration(Table 6), the effect of carbopol was still not a synergistic one. Thisresult could be attributed to that the amount of STF that was mixedwith the formulae to simulate physiological conditions was notsufficient to increase the pH sufficiently to properly neutralize carbopolto induce gelation. When carbopol become neutralized to a pHenvironment of 5.5–9.0, the added ions preferentially enter thenegatively-charged polymer backbone, which leads to an imbalancein osmotic pressure and water is drawn into the macromolecularstructure, consequently, they swell and expand up to 1000 times theiroriginal volume (Gutowski, 2010). The pH increased after addition ofSTF only to 5.8, which indicates only partial neutralization of carbopol.Furthermore, the increase in carbopol concentration caused a decreasein the viscosity of the formed in situ gels. This finding is probablyrelated to the associated decrease of pH value below 5.5 due to theacidic nature of carbopol. At pH values below the pKa value of carbopol(5.5) no sol to gel transition occurs as the molecular structure is not yetswollen enough to be space filling (Gutowski, 2010). Other researchersmanaged this problem by partial neutralization of carbopol by adjustingpH of the formula near physiological value (Kumar et al., 2012).However, this could not be employed as it would have affected stabilityof AZA. Interestingly, at highest carbopol concentration (F10) theviscosity was even lower than F1, which contains only gellan gum,taking into consideration that a previous work done by Picone andCunha (2011) showed that at lower pH values the gel structure of gellanbecame denser and more compact. This result indicates that thecarbopol molecules, which are probably coiled at pH 5, hinder thegellan double helices to join junction zones.

From the above findings, it could be concluded that the combinationof xanthan gum, HPMC or carbopol did not exert a remarkable effect onin situ gelation and they would therefore serve as viscosity enhancerswith the exception of carbopol formulae at concentrations> 0.2%.

3.4.4. Gel strength determinationIn the development of in situ gel, appropriate gel strength of the

formulation is important. The formulation must be administered easilyas drops and must have sufficient strength to prevent rapid drainage ofthe formulations. Mahajan and Gattani (2010) who used similarapparatus and conditions to those applied in the present study statedthat the gel strength values between 25 and 50 s were consideredappropriate. Too low gel strength (< 25 s) indicates weak gel struc-

tures that may not retain their integrity and may erode rapidly, whilevery stiff gels (> 50 s) may cause discomfort to the mucosal surfaces(Mahajan et al., 2012). All formulae showed optimum gel strength asthe determined gel strength values ranged between 29 and 41 which arewithin the acceptable range (Table 6). Furthermore, the gel strengthvalues were found to be closely related to the viscosity of the formedgels as an increase of gel viscosity was accompanied by an increase ofgel strength. This is in accordance with the degree of crosslinking of thein situ gel. Gels with greater number of crosslinks per unit volume showhigher gel strength values and hence higher viscosities (Draget et al.,1993).

3.4.5. Mucoadhesive strengthThe mucoadhesive force is an important physicochemical parameter

for in situ forming ophthalmic gels because it prevents the formulationfrom rapid drainage and hence lengthens its precorneal residence time.The degree of bioadhesion depends on type and amount of polymer,degree of hydration, polymer chain length, molecular weight ofpolymer, degree of interpenetration of polymer chains in addition totype of bonding between polymer chains and the glycoprotein chainsattached to the epithelial cells of mucus membranes (Andrews et al.,2009). The bonding of mucoadhesive polymers to mucosal membraneoccurs primarily through hydrophobic interactions, hydrogen bonding,van der Waals bonds as well as ionic interactions (Woodley, 2001).

From Table 6 it is to be noticed that, the addition of mucoadhesivepolymers reinforced the mucoadhesive force of gellan gum formula.The mucoadhesive polymers could be arranged according to theirmucoadhesive enhancing effect at 0.2% concentration as follows:carbopol 940 > xanthan gum > HPMC. With exception of carbopolit was found that increasing the concentration of the mucoadhesivepolymers in the formulations significantly increased the mucoadhesiveforce. On contrary, the adhesive strength of carbopol was not signifi-cantly affected (p < 0.05) when concentration was increased to 0.4%and even decreased when its concentration was increased to reach0.6%. Thus at concentrations 0.4 and 0.6% the rank of mucoadhesivestrength changed to xanthan > carbopol > HPMC. Anionic polyelec-trolytes have been found to form stronger adhesion with the glycopro-tein chains of the mucus layer when compared with neutral polymers asthe presence of charged functional groups in the polymer chain has amarked effect on the strength of the bioadhesion (Peppas and Buri,1985). This could explain the relatively low mucoadhesive strength ofnon-ionic HPMC compared to anionic xanthan and carbopol. Themucoadhesive strength of xanthan gum could be attributed to physicalentanglement of polymer chains in addition to van der Waals forces andhydrogen bonding with mucin. The good mucoadhesive performance ofxanthan is probably attributed also to its high molecular weight, whichwas indicated by the high viscosity of its formulations. It is reportedthat, the mucoadhesive strength of a polymer increases with increasingmolecular weight due to deeper interpenetration of polymer and mucuschains in addition to the increase in number of polar functional groupson the molecular chain capable of forming hydrogen bonding withmucin (Jiménez-castellanos et al., 1993). However, its mucoadhesion islower than that of carbopol at 0.2%, which may be a result of the sterichindrance of the side chains that interfere with binding to the chargedmucin groups as previously proposed by Ceulemans et al. (2002). At0.2% polymer concentration carbopol was found to exhibit the highestmucoadhesion. This is probably due to hydrogen bonding betweenmucin and the abundant carboxylic acid groups on the molecular chain(Kaur et al., 2002). The decrease in mucoadhesion strength by furtherincrease in carbopol concentration is probably attributed to theaccompanied lowering of the pH of the formulation. At pH valuesbelow 5.5 the dissociation of the carboxylic groups present on thepolymeric chain decreases and the molecules become less swollen andmore coiled, which in turn will decrease the levels of entanglementwithin the mucus layer and reduces the available hydrogen bondinggroups that could combine with mucin (Zhu et al., 2013). Moreover, a

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study made by (Rakde et al., 2015) showed that the mucoadhesivestrength of gellan gum increases as pH increases in the acidic medium(up to pH value of 6). Thus, the increase in concentration of acidiccarbopol lowers the pH of the formulation, which in turn lowers themucoadhesive strength of gellan.

The in situ gelling formulations which contain 0.3% gellan gum inaddition to 0.2% xanthan gum, HPMC and carbopol 940, respectively(F2, F5 and F8) were selected for further investigations. They wereselected for having acceptable physicochemical properties and theincrease in polymer concentration to 0.4 and 0.6% did not favorablyalter the characteristics of the formulae, taking into consideration thatformulations with higher polymer concentration are more susceptibleto induce ocular irritation.

3.4.6. Transmission electron microscope (TEM)The selected in situ gelling formulae (F2, F5 and F8) and the

corresponding NE were subjected to examination under TEM in order tostudy the effect of addition of different polymers on the morphology ofthe NE (Fig. 2).

It is clear that the developed NE possessed spherical and discretedroplets As observed from TEM images of in situ gelling systems theemulsified droplets retained their spherical structure with mild loss ofsmooth spherical boundaries. Although the PDI values recorded forthese systems were all lower than 0.3, which indicates a uniformdroplet size distribution, the figures show droplets of larger sizes amongsmaller ones. Probably because the PDI calculation takes into accountall droplets in the medium, while the figure captures only a very smallpopulation of the system.

3.4.7. Droplet size, polydispersity index and zeta potentialFrom Table 7 it could be noticed that the droplets retained their size

after the incorporation of in situ gelling polymers having high degree ofhomogeneity, which is indicated by the small polydispersity index(smaller than 0.3). However, due to the anionic nature of gellan gum agreat reduction of zeta potential was observed for in situ gelling systems(Table 7). The combination of the different mucoadhesive polymersalso affected the zeta potential significantly. It is to be noted that, theincorporation of the neutral polymer, HPMC, elevated the zeta potentialto a certain extent, while the combination of anionic xanthan gumfurther shifted the zeta towards higher negative values. Althoughcarbopol 940 is also an anionic polymer with high charge density, itscombination with gellan gum resulted in a slight elevation in zetapotential. This result supports the belief that the carboxylic groups arenot sufficiently ionized at pH 5.8.

3.4.8. Degree of transparencyThe percentage transmittance (% T) of the formula reflects the

degree of its transparency which in turn could be a measure of thedegree of interference with vision upon ocular application. The % T ofthe selected in situ NE gels is shown in Table 7. It is to be noticed thatall NE based in situ gelling formulae showed high degree of transpar-ency in the sol state. This indicates that the very small NE droplets andthe randomly scattered polymer molecules experience a low degree oflight scattering. However, upon gelation a reduction of about 15% intransparency of the formulae could be observed. It was reported by(Mao et al., 1998) that, when a polymer solution becomes a physicallycrosslinked gel with orderly packed junction zones, the intensity of thescattered light increases, which reduces the clarity of the gel. This resultwas in accordance with the viscosity measurements of the in situ gels.The increased viscosity of gellan in situ gel upon combination ofxanthan, HPMC and carbopol was accompanied by a respectivereduction in gel transparency due to increased degree of crosslinking

Fig. 2. Transmission electron micrographs of a) nanoemulsion (NE6) b) nanoemulsion in situ gel gellan/xanthan (F2) c) nanoemulsion in situ gel gellan/HPMC (F5) d) nanoemulsion insitu gel gellan/carbopol (F8).

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and increased length of junction zones.

3.5. In vitro drug release study

In vitro release studies were performed to investigate the effect ofgellan gum alone (F1) and in combination with different polymers (F2,F5 and F8) in NE based in situ gelling formulations on the release ofdrug with respect to the corresponding NE. In order to comparebetween different formulations, the percent of AZA released versustime was plotted (Fig. 3) and the area under the curve (AUC0-8h) as wellas the percentage of drug released after 2 and 8 h were determined(Table 8). It could be observed that, the drug release decreased by morethan two-folds from all NE based in situ gelling systems in comparisonto the corresponding NE. The NE showed a 100% drug release after 4 h,while at 8 h about 40% (F2)–60% (F1, F5 and F8) of AZA was releasedfrom in situ gelling systems. By applying one way ANOVA, it could beseen that all in situ gel formulations were significantly different(p < 0.0001) from the NE in terms of the three parameters listed(AUC0–8 h, % AZA released after 2 h and 8 h). This result indicates thatthe increased viscosity and crosslinking density of the gelled formula-tions effectively retarded the drug release. Among the in situ gellingformulae, F2 that contains xanthan gum showed the slowest releaseprofile, although it did not possess the highest viscosity. This finding

may be attributed to the semi-rigid structure of xanthan network(Nagendra et al., 2014). The fact that xanthan gum is an ion activatedin situ gelling polymer may be another reason. Although its additiondid not show great effect on in situ gelation upon mixing with STF atratio 25:7, the contact with STF for long time via diffusion ofelectrolytes may have reserved the complex network due to intermo-lecular associations (Sworn, 2009). By comparing (F5) and (F8) with(F1) to study the effect of addition of HPMC and carbopol 940,respectively to gellan gum on the extent of release of AZA, it wasfound that they have similar extent of release, as AUC0-8h of both F5 andF8 was not significantly (p > 0.05) different from AUC0–8 h of F1.

However, F8 containing carbopol 940 showed significant differencefrom F1 with respect to percentage AZA released after 2 h (p < 0.05)and non-significant difference after 8 h (p > 0.05). This result leads tothe conclusion that carbopol initially influences the release of AZA butthis effect disappears after about 3 h. This result was found by otherresearchers (Kumar et al., 2012) who reported that, formulations ofcarbopol disrupt and lose their integrity rapidly and release the drugwithin few hours.

Despite that F5 showed a non-significant difference from F1 in termsof AUC and percent drug released after 2 h, it showed significantlylower percent drug released at the end of the study (8 h). Such resultindicates that HPMC maintains the integrity of the gel for long timealthough its effect on sustainment was not significant at the beginningof the release experiment. This finding could be explained in the light ofa study made by Joshi (2011) who concluded that the thermo-reversiblebehavior of HPMC is practically affected by the nature of salt additives.He found that the presence of some salts (especially anion parts) likeCl− in the gelation medium causes both a decrease in gelationtemperature as well as an increase in gel strength of HPMC polymer.This type of anions facilitates the hydrophobic associations of methylsubstitutions between the molecules through a strong electrostaticorientation of water molecules which makes water structure moreordered towards the anion. This effect was detected at the end of thestudy after diffusion of Cl− from STF and its interaction with HPMC togive a more strengthened gel structure that slowed down the drugrelease. Probably, this interaction is a slow process thus its effect wasonly detected at the end of the study.

3.5.1. Kinetic study for drug releaseThe results of applying various kinetic models to the release data of

AZA from different formulations revealed that the drug release from allof NE based in situ gels followed Higuchi diffusion model.

3.6. Thermodynamic stability studies

Accelerated stability testing was carried out for the selected NE insitu gels (F2, F5 and F8) at 40, 25 and 4 °C for 3 months. Samples wereexamined every month for physical appearance, drug content, pH andviscosity. It was found that time and temperature did not affect physicalappearance of F2 and F5 as they remained clear liquids at differentstorage temperatures for 3 months. However, F8 showed partialprecipitation of the drug upon storage at all the studied temperatureswhich indicated its instability. This may be due to incompatibilitybetween carbopol and AZA. Carbopol, being a poly-acrylic acid

Table 7Characterization of selected NE based in situ gels (mean ± SD, n = 3).

Formula Droplet diameter (nm) Zeta potential (mV) Polydispersity index (PDI) Transmittance (%)

Sol Gel

F1 13.41 ± 0.16 −19.7 ± 0.7 0.24 ± 0.035 99.7 ± 0.11 86.7 ± 0.03F2 13.26 ± 0.26 −22.3 ± 2.7 0.18 ± 0.058 96.3 ± 0.14 82.8 ± 0.12F5 13.34 ± 0.49 −13.7 ± 0.0 0.25 ± 0.026 98.8 ± 0.13 84.5 ± 0.09F8 13.01 ± 0.13 −15.6 ± 0.3 0.27 ± 0.13 95.6 ± 0.14 80.5 ± 0.11

Fig. 3. Percent of drug released from nanoemulsion (NE6), nanoemulsion in situ gelgellan (F1), nanoemulsion in situ gel gellan/xanthan (F2), nanoemulsion in situ gelgellan/HPMC (F5) and nanoemulsion in situ gel gellan/carbopol (F8) (n = 3).

Table 8In vitro release study for NE based in situ gels (mean ± SD, n = 3).

Formula AUC0–8 ha % AZA released after

2 h% AZA released after 8 h

NE6 375.91 ± 13.79 80.18 ± 2.40 99.11 ± 1.57F1 154.98 ± 1.02 29.56 ± 0.89 61.49 ± 1.70F2 93.63 ± 5.95 18.48 ± 1.57 37.9 ± 1.21F5 140.19 ± 4.611 27.34 ± 1.08 53.87 ± 1.70F8 140.61 ± 5.07 25.43 ± 1.27 58.32 ± 1.08

a Area under percentage drug released versus time curve from 0 to 8 h.

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derivative showed incompatibility with other drugs. Jain et al. (2008)and Bilensoy et al. (2006) reported incompatibilities between carbopoland ciprofloxacin hydrochloride and clotrimazole, respectively, whichresulted in drug separation. With respect to drug content, the observedresult was in accordance with the former one, where F2 and F5 showedalmost constant drug content till the end of the third month, while F8showed a marked decrease in drug content from 99.8% to 80.2% afterthe first month due to precipitation of the drug. Based on these resultsthe stability study was not continued for F8 after the first month and theformula was excluded from further studies. With respect to pH andviscosity, F2 and F5 did not show remarkable changes during the timeof study (data not shown).

3.7. Ocular irritation studies

The examination of the conjunctiva and other parts of the eye wasperformed by external observation under proper illumination afterinstillation of F2, F5 and 0.9% NaCl into rabbits' eyes. The total Draizesum of scores was calculated for each time point to evaluate possibleocular irritation of the developed formulae (Table 9). The studyrevealed no alteration in conjunctiva and other parts of rabbit's eye.Furthermore, no significant conjunctival discharge, redness or chemosiswere observed in any of the rabbits after instillation of F2 and F5 withthe exception of slight redness and lachrymation, which disappeared

after 30 min.

3.8. Therapeutic efficacy study (in vivo pharmacodynamic study)

An in vivo pharmacodynamic study on selected NE based in situ gelformulae (F2 and F5 after exclusion of F8) in comparison with the

Table 9Sum of mean scores for grading the severity of ocular irritation (Iirr) after ocularapplication of the developed AZA loaded NE based in situ gels according to modifiedDraize method (Baeyens et al., 2002).

Formula Total (Iirr) at different observing times (min)

5 10 15 30 60 120

F2 0.33 0.67 1 0.33 0 0F5 0.33 0.67 0.67 0.67 0 00.9% NaCl 0 0 0 0 0 0

Fig. 4. Change of IOP values with time in the treated glaucomatous eye in comparison to the established baseline of the same glaucomatous eye before treatment for a) nanoemulsion insitu gel gellan/xanthan (F2), b) nanoemulsion in situ gel gellan/carbopol (F5), c) Azopt® eye drops and d) Cidamex® tablets (n = 3).

Fig. 5. Percentage decrease in IOP after topical application of nanoemulsion in situ gelgellan/xanthan (F2), nanoemulsion in situ gel gellan/carbopol (F5), Azopt® eye drops andCidamex® tablets (n = 3).

Table 10Pharmacodynamic study developed AZA loaded NE based in situ gels, Azopt® eye dropsand Cidamex® tablets.

Formula Maximum decrease in IOP (%) tmax (h) AUC0–10 (%·h)

F2 35.25 ± 3.69 2.67 ± 0.58 189.15 ± 10.18F5 32.35 ± 1.92 2.0 ± 0.87 146 ± 17.52Azopt 33.84 ± 1.42 1.5 ± 0.0 82.51 ± 7.53Cidamex 24.01 ± 0.02 1.5 ± 0.0 79.77 ± 7.58

IOP: intraocular pressure, AUC0–10: area under the curve of percentage decrease in IOPversus time from 0 to 10 h.

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commercial eye drops (Azopt®) and the commercial oral tablet(Cidamex®) over a period of 10 h was performed. Azopt® eye drops,which contains 1% brinzolamide as carbonic anhydrase inhibitor, wasselected to be the topical control for the study because there is notopical AZA formulation available in either local or internationalmarkets. Taking into consideration that brinzolamide is more intrinsi-cally permeable than AZA (Kaur et al., 2002), makes the comparison aninteresting challenge. Cidamex® tablet was selected as the secondcontrol in the study as it represents the oral AZA formulation in themarket. Cidamex® tablets were used to emphasize the differences in thetherapeutic efficacy between the developed topical 1% AZA NE based insitu gels and the per-oral tablets of AZA in a dose equivalent to 9 mgwhich is the calculated dose for rabbits of an average weight of 2.5 kg.

The effect of F2 and F5 in situ gels as well as Azopt® eye drops andCidamex® oral tablets on IOP is shown in Fig. 4, which illustrates thechange of IOP values in the treated glaucomatous eye with time incomparison to the established baseline of the same glaucomatous eyebefore treatment. Fig. 5, which presents the percent decrease of IOP inglaucomatous eye versus time of various selected formulations andmarket products, was used to calculate the following pharmacodynamicparameters: AUC, tmax, MRT and maximum percentage decrease in IOP.These parameters (Table 10) were used to facilitate the comparisonamong different formulations.

It is evident that both F2 and F5 showed a prolonged IOP loweringactivity over Azopt® and Cidamex®. Considering the parameter[AUC]0 → 10, it is noticed that its value ranked in the following order:F2 > F5 > Azopt® > Cidamex®. By applying ANOVA statistical test,the differences were statistically significant (p > 0.05) with theexception of Azopt® and Cidamex®, where variation among them wasconsidered non-significant (p < 0.05).

In the view of the above results, it could be seen that both AZA-NEbased in situ gelling formulations had higher therapeutic efficacy thanboth Azopt® eye drops and Cidamex® tablet where F2 showed thehighest therapeutic activity with AUC 2.29 and 2.37 times higher thanthat of Azopt® drops and Cidamex® tablet, respectively followed by F5which showed AUC 1.77 and 1.83 times higher than that of Azopt®drops and Cidamex® tablet, respectively.

The poor wettability and relatively fast clearance from the pre-ocular surface lowered significantly the therapeutic efficacy of brinzo-lamide from Azopt® eye drops. Moreover, the non-selective inhibitionof carbonic anhydrase enzyme, that is distributed all over the body, byAZA after per-oral administration of Cidamex® tablet led to decrease inits ocular therapeutic efficacy taking into consideration that theadministered tablet dose was equivalent to 9 mg AZA while the dropof NE based in situ gel contained only about 0.5 mg of AZA.

Higher AUC obtained by in situ gelling formulation is probablybecause of the greater penetration of the drug from NEs due to thepresence of surfactants and cosurfactants which increase the cornealpermeability, thereby increasing drug uptake. In other words, NEs actas penetration enhancers by disrupting tight junctional complexes tofacilitate corneal drug delivery (Kaur and Smitha, 2002). Furthermore,the submicron droplets could penetrate into the corneal epithelium cellsby endocytosis (Yu et al., 1993). In addition, the increase in residencetime on corneal epithelial layer because of in situ gelation upon contactwith cations of tear fluid as well as the increase in corneal wettabilitywould enable better transcorneal drug penetration to the ocularinternal tissues.

With respect to the duration of IOP reduction, it is evident fromFig. 4 and Fig. 5 that both F2 and F5 showed a prolonged effectcompared to Azopt® or Cidamex®. The prolonged IOP lowering effectexhibited by F2 and F5 could be correlated to the increase in the ocularresidence time due to in situ gelation of gellan gum and mucoadhesiveand viscosity enhancement effect of the combined polymers.

No change in IOP was observed in the untreated eye during thecourse of measurement in any of the two in situ gelling formulations,which indicates that F2 and F5 exerted an inhibition of carbonic

anhydrase enzyme only within the treated eye and that the observedIOP lowering activity is not because of any systemic absorptionfollowed by subsequent redistribution.

4. Conclusion

In the present study AZA loaded nanoemulsion composed of peanutoil, a surfactant mixture of tween 80 and cremophor EL in addition totranscutol P as cosurfactant was selected to formulate nanoemulsionbased ion induced in situ gels for ocular administration. Gellan gum in aconcentration of 0.3% w/w showed optimum gelation when mixed withsimulated tear fluid. The incorporation of xanthan gum, HPMC andcarbopol 940 did not exert a remarkable effect on in situ gelation;however, their addition significantly reinforced the mucoadhesive forceof gellan gum in situ gel. The acidic nature of carbopol lowered the pHof the formulation at increased polymer concentration, which resultedin reduced ionization of carboxyl groups and increased coiling of thepolymer molecules; this was reflected in marked reduction in viscosity,gel strength and mucoadhesion. The gellan/carbopol formulation alsoshowed drug separation in accelerated stability testing at all studiedstorage temperatures. The gellan/xanthan formula on the other handshowed good stability, could effectively slow down the drug release andshowed superiority in lowering intraocular pressure for a prolongedtime with respect to marketed brinzolamide eye drops (Azopt®) andoral acetazolamide tablets (Cidamex®). This formulation thus presents apromising topically applied dosage form for the treatment of glaucomawith reduced systemic side effects of AZA due to the local inhibition ofcarbonic anhydrase. It also exerted a prolonged therapeutic effect,which would improve patient compliance due to less frequent admin-istration.

Declaration of interest

The authors report no conflicts of interest.

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