Research Article

Non-ionic Surfactant Based In Situ Forming Vesicles as Controlled ParenteralDelivery Systems

Hussein O. Ammar,1 Magdy Ibrahim,2 Azza A. Mahmoud,1,3,4 Rehab N. Shamma,2 and Nada M. El Hoffy1

Received 19 July 2017; accepted 27 September 2017; published online 6 November 2017

Abstract. Non-ionic surfactant (NIS) based in situ forming vesicles (ISVs) present anaffordable alternative to the traditional systems for the parenteral control of drug release. Inthis work, NIS based ISVs encapsulating tenoxicam were prepared using the emulsionmethod. Tenoxicam-loaded ISVs were prepared using a 22.31 full factorial experimentaldesign, where three factors were evaluated as independent variables; type of NIS (A), molarratio of NIS to Tween®80 (B), and phase ratio of the internal ethyl acetate to the externalCaptex® oil phase (C). Percentage drug released after 1 h, particle size of the obtainedvesicles and mean dissolution time were chosen as the dependent variables. Selectedformulation was subjected to morphological investigation, injectability, viscositymeasurements, and solid state characterization. Optimum formulation showed sphericalnano-vesicles in the size of 379.08 nm with an initial drug release of 37.32% in the first hourfollowed by a sustained drug release pattern for 6 days. DSC analysis of the optimizedformulation confirmed the presence of the drug in an amorphous form with the nano-vesicles.Biological evaluation of the selected formulation was performed on New Zealand rabbitsby IM injection. The prepared ISVs exhibited a 45- and 28-fold larger AUC and MRT values,respectively, compared to those of the drug suspension. The obtained findings boost the useof ISVs for the treatment of many chronic inflammatory conditions.

KEY WORDS: in situ forming vesicles; non-ionic surfactants; intramuscular; emulsion; anti-inflammatory.

INTRODUCTION

Parenteral controlled release systems present an efficientapproach for treatment of many diseases. It offers manyadvantages including extended drug release period, constantdrug plasma concentration, localized delivery of the drug, andimproved bioavailability (1,2). Intramuscular (IM) injection isthe major administration route for parenteral controlledrelease systems.

The administration of drugs (hydrophilic/hydrophobic)encapsulated in vesicular systems is an innovative mean of

delivering drugs in a controlled manner (3–8). Vesicularsystems such as liposomes and niosomes are common carriersto encapsulate the drug molecules (9). Unfortunately, suchvesicular systems suffer from limited stability, encapsulation,and scaling up problems which arouse the need for develop-ing new vesicular systems which can overcome such obstacles.

In situ forming vesicular systems offer superior proper-ties over the classical implants. For example, the applicationof these formulations is less painful compared to the implantsystems, and the manufacture of such systems is less complex(10). In situ forming systems consist of a solution ordispersion of a drug in a biodegradable polymer. After beinginjected, the polymer solidifies and generates a solid matrix,which controls the drug release. On the other hand, in situforming implants involve viscous polymeric solution, whichincreases the pain upon injection. To overcome this problem,in situ forming microparticle systems were developed. Thesesystems are based on an emulsion with an internal organicphase of polymeric solution and a continuous oil phase. Onceinjected, the polymeric solution droplets solidify due todiffusion of the organic solvent, resulting in the formation ofin situ forming microparticles (1).

In situ forming particles based on PLGA are promisingdrug delivery systems (11–13), but unfortunately, PLGA is an

Electronic supplementary material The online version of this article(https://doi.org/10.1208/s12249-017-0897-8) contains supplementarymaterial, which is available to authorized users.1 Department of Pharmaceutics and Pharmaceutical Technology,Faculty of Pharmaceutical Sciences and Pharmaceutical Industries,Future University in Egypt, End of 90th street, Fifth settlement,New Cairo, Egypt.

2 Department of Pharmaceutics and Industrial Pharmacy, Faculty ofPharmacy, Cairo University, Kasr El-Aini Street, Cairo, Egypt.

3 Department of Pharmaceutical Technology, National ResearchCenter, Cairo, Egypt.

4 To whom correspondence should be addressed. (e-mail:azza.ahmed@fue.edu.eg)

AAPS PharmSciTech, Vol. 19, No. 3, April 2018 (# 2017)DOI: 10.1208/s12249-017-0897-8

1001 1530-9932/18/0300-1001/0 # 2017 American Association of Pharmaceutical Scientists

expensive polymer. Therefore, there is a need to formulate anew in situ forming vesicular system based on less expensivematerials that can provide controllable drug release. Non-ionic surfactants based vesicular systems have the ability toimprove the solubility of poorly water soluble drugs and todeliver the drug in a controlled rate (3,14–20). Non-ionicsurfactants offer many advantages over other ionic surfac-tants; they are nontoxic, non-irritant, in addition to beinginert. They are commonly used in the formulation of differentoral and parenteral pharmaceutical preparations (21).

Tenoxicam (TX) is a member of the oxicam family(22,23) characterized by potent anti-inflammatory (24),antipyretic, and analgesic effects (25). TX is practicallyinsoluble in water and very slightly soluble in ethanol. TXside effects’ resemble other NSAIDs, influencing GITcausing GI ulceration (26) and may lead to renal failure orbleeding (27). Encapsulating TX in parenteral controlledISVs would offer a beneficial way of reducing the dosingfrequency, and the GI disorders associated with the oraladministration especially in the treatment of post-operativepain, and different chronic inflammatory conditions as acutegout, rheumatic diseases, and osteoarthritis which would bemore convenient for the patient, despite the pain associatedwith the IM injection.

In this work, novel parenteral controlled, non-ionicsurfactant based, in situ forming vesicles (ISVs) encapsulatingTX were prepared using the emulsion method. TX-loadedISVs were prepared according to a 22.31 full factorialexperimental design in order to investigate the jointinfluence of formulation variables using Design-Expert®software. The prepared systems were characterized for thein vitro drug release profiles and particle size of the obtainedvesicles. The formulation showing the optimum releaseprofile was selected for further investigations includingmorphological examination of the obtained vesicles usingtransmission electron microscopy, injectability, viscositymeasurement, and solid state characterization of the drugusing differential scanning calorimetry. Biological evaluationof the selected formulation was performed in a group of sixNew Zealand rabbits and compared to those of the drugsuspension. The pharmacokinetic parameters for the twotested formulations were calculated.

MATERIALS AND METHODS

Materials

Tenoxicam (TX) was a kind gift from EIPICO Co.,Egypt. Sorbitan monooleate (Span® 80), polyethylenesorbitan monooleate (Tween® 80), and lornoxicam werepurchased from Sigma-Aldrich Chemie GmbH, Steinheim,Germany. Captex® (355 EP/NF) (caprylic/capric triglyceride)was kindly donated from ABITEC Corporation, Janesville,USA. Brij 52 and acetonitrile (HPLC grade) were purchasedfrom Sigma-Aldrich, USA. Ethyl acetate was purchased fromPharmasolve, ISP customer service GmbH, Köln, Germany.Di-sodium hydrogen orthophosphate anhydrous (min assay-acidimetric 98%) and sodium di-hydrogen orthophosphate-1-hydrate (min assay 98%) were purchased from ADWIC,Egypt.

Preparation of TX-loaded ISVs

Accurately weighted amount of the NIS (2%) (Span®60/Brij® 52 and Tween® 80) was dissolved in ethyl acetate inthe desired molar ratios by sonication (water bath sonicator;Soniclean 120T, Australia) until a clear solution was obtained.Accurately weighed amount of TX was then added into theNIS-ethyl acetate solution and well dispersed by sonication.The ISVs were prepared by emulsifying the NIS-drug mixtureinto the oil phase (Captex® stabilized with 4% w/v Tween®80 representing the external phase) at different phase ratioswith sonication for 10 min at room temperature. Theconcentration of TX in the final formulation was keptconstant at 1 mg/mL.

In Vitro Drug Release Studies

In vitro drug release studies of TX from the preparedformulations were carried out using the dialysis bag method(28). Exactly, 0.5 mL TX-loaded ISVs was injected into thedialysis bag (cellulose membrane dialysis bag of 6-cm longand 2.2-cm wide, with a molecular weight cut off 12–14,000 Da, Sigma Chemical Company, USA) acting as thedonor compartment. The dialysis bag was then placed into10 mL phosphate buffer (pH 7.4), representing the receptorcompartment, with the temperature maintained at 37 ± 0.5°Cin an incubation shaker (GFL. type 3032, USA) stirring at arate of 100 stroke per minute. At pre-determined timeintervals, 3 mL of the receptor medium were taken andreplaced by fresh medium. The withdrawn samples wereanalyzed spectrophotometrically for drug content at 369 nm.

Percentages of mean cumulative drug released wereplotted against time, and the release data were kineticallyanalyzed using different kinetic models to determine themechanism of drug release from the prepared formulations,and release parameters were calculated to compare theinvestigated formulations. The investigated release parame-ters were cumulative percentage of drug released after 1 h(Q1) and mean dissolution time (MDT), which is a measureof the retarding ability of the formulation against drugrelease.

The kinetic analysis of TX release from ISVs wasdetermined by using linear regression analysis in order tofind the best fit of the release data to equation representing:zero (29) and the Higuchi diffusion release model (30) as wellas applying the Korsmeyer-Peppas equation (31–33).

Particle Size Determination

The prepared ISVs were treated as explained underin vitro drug release study; the dialysis bags were opened after24 h, and the obtained emulsions were centrifuged at15,000 rpm at 4°C for 15 min (Sigma 3-30K, Model 3-30K,Germany) followed by removal of the oily phase. Theobtained vesicles were separated, re-suspended in 1 mLphosphate buffer solution (pH 7.4), and examined for particlesize. The average particle size and size distribution of theobtained vesicles were determined by dynamic light scattering( D L S ) a n a l y s i s u s i n g Z e t a S i z e r N a n o Z S(MalvernInstruments, UK); n = 3 ± SD.

1002 Ammar et al.

Statistical Analysis of the Experimental Design

TX-loaded ISVs were optimized using a 22.31 fullfactorial experimental design in order to investigate thejoint influence of formulation variables using Design-Expert® software (Stat-Ease, USA). In the 22.31 design,three factors were evaluated as independent variables; twoat two levels, and the third at three levels. Experimental trialswere performed at all 12 possible combinations, where threefactors were evaluated as independent variables: (A) type ofNIS (Span®60 or Brij®52), (B) molar ratio of NIS to Tween®80 (8:1, 4:1, and 2:1), and (C) phase ratio of the internal ethylacetate to the external Captex® oil phase (1:4 and 1:2).Percentage drug released after 1 h (Q1; with target value lessthan 40%), the mean PS (with target value less than 1000 nm)of the obtained vesicles, and the MDT were chosen as thedependent variables.

Transmission Electron Microscopy

Selected preparation was treated as explained underin vitro drug release study, but the dialysis bags were openedafter 24 h of immersion in the phosphate buffer saline toobtain the ISVs, and the obtained systems were centrifuged at15,000 rpm at 4°C for 15 min (Sigma 3-30K, Model 3-30K,Germany) followed by removal of the oily phase. Theobtained vesicles were separated, re-suspended in 1 mLphosphate buffer solution (pH 7.4), and examined formorphology using transmission electron microscopy (TEM).

The shape of vesicles of the selected formulation wascharacterized by TEM working at an accelerating voltage of80 kV. A drop of the formed vesicles was placed on a carbon-coated copper grid and left to adhere onto the carbonsubstrate for about 2 min. A drop of 2% w/v phosphotungsticacid solution was added onto the carbon grid. Finally, thesamples were air dried and the thin film of stained ISVs wasexamined.

Differential Scanning Calorimetry

Selected preparation was treated as explained underin vitro drug release study, but the dialysis bags were openedafter 24 h of immersion in the phosphate buffer saline toobtain the ISVs, which were further freeze-dried (Christfreeze dryer, ALPHA 2-4 LD plus, Germany), and examinedby differential scanning calorimetry (DSC) (DSC-50,Schimadzu, Kyoto, Japan). DSC thermograms for the indi-vidual components (Span® 60 and TX) were also investigated.An accurately measured amount of each sample (5 mg) wasweighed into an aluminum pan and analyzed as sealed withpinholes, and an empty aluminum pan was used as reference.The process was operated at a heating rate of 10°C/min andemployed over temperature range from 20 to 300°C underinert nitrogen flow (25 mL/min).

Viscosity Measurements

The viscosity of the selected ISVs system was measuredby a rheometer (Brookfield DV3THB cone/plate rheometer,spindle CPE-40 and RheocalcT software, v 1.1.13 software) at25°C ± 0.2 (PolyScience model 9006, USA). The viscosity of

ISVs preparation was measured by a plate and coneconfiguration 20-mm diameter/4° angle with a fixed shearrate (1/s).

The flow behavior was studied according to Farrow’sequation (34): log D = N log S − log η.

where, D is the shear rate (sec−1), S is the shear stress(Pa), N is Farrow’s constant, and η is the viscosity (Pa.s).

N is the slope of the plot of log D against log S, whichindicates the deviation from Newtonian flow. When N is lessthan 1, it indicates dilatants flow (shear rate thickening). If Nis greater than 1, it indicates pseudoplastic or plastic flow(shear rate thinning) (34).

Injectability

In order to evaluate the ease of injection, the selectedformulation was subjected to injectability testing and com-pared with that of a market oily injection, Betolvex™, andwas assessed using home-based equipment designed for thispurpose (35). The designed equipment was similar to thatpreviously described by Leroux et al. and Lacout et al. (36,37)but with some modifications. A 5-mL syringe attached to a19-gauge needle was filled with constant volume of the testedformulation, and then the filled syringe was fixed to a rubbertube ending with an air pump. Air was forced from the airpump on the solution surface, and the applied pressure on thesolution surface was maintained constant (at 70 mmHg) usinga sphygmomanometer (38). The time required to release a1-mL sample was recorded, and the values of the flow rate(mL/min) were compared to give an indication for theformulae injectability (39).

In Vivo Evaluation of Selected TX-loaded ISVs

Study Design

Six male New Zealand rabbits (mean body weight of3.00 ± 0.25 Kg) were randomly distributed into two groups, ofequal number. The rabbits were kept in individual cagesunder well-defined and standardized conditions (humidity-and temperature-controlled room; 12-h light and 12-h darkcycle). Rabbits were fed with a standard rabbit diet with freeaccess to water. The rabbits were fasted for 24 h prior to andduring the pharmacokinetic study. All animal procedureswere performed according to the protocols reviewed andapproved by the Research Ethics Committee of Faculty ofPharmacy, Cairo University.

The selected formulation was compared to TX suspen-sion in phosphate buffer (pH 7.4) with equivalent drugconcentration. All formulations were sterilized using gammaradiation at a dose of 10 kGy (1.88 kGy/h). Rabbits wererandomly divided into two groups, each containing threerabbits. The study was performed using a simple randomizedcross-over design where the first group received 1.5 mL of theselected ISV formulation by IM injection while the secondgroup received 1.5 mL TX suspension by IM injection, bothequivalent to 1 mg/mL. After a washout period of 2 weeks,the two groups were crossed-over. After IM injection of thetested formulations, the rabbits were monitored closely forsigns of local injection-site reactions (redness, itching, orswelling).

1003Non-ionic Surfactant Based In Situ Forming Vesicles

At the pre-determined time intervals, blood sampleswere withdrawn from the marginal ear vein into heparinizedvial. Serum was separated by centrifugation for 10 min at3900×g and frozen at − 30°C for future measurement (1). Theconcentration of the drug in the serum was assessed using amodified UPLC method.

Chromatographic Conditions

Blood samples were analyzed for TX adopting amodified sensitive, selective, and accurate isocratic UPLCmethod developed and validated before the study. Plasmasamples were spiked with lornoxicam (as an internal stan-dard) and mixed with 300 μL of acetonitrile-perchloric acidsolution (2:1 v/v), followed by centrifugation for 10 min at3500 rpm. The supernatant was separated, filtered through0.45-μm Millipore® filter, and then injected to the UPLCcolumn for analysis (Agilent UHPLC, 1290 Infinity LC,binary pump, autosampler, multicolumn thermostat, USA).

The isocratic mobile phase was composed of ammoniumacetate buffer (pH 2.7) and acetonitrile (72:28 v/v). Themobile phase was filtered and degassed. The flow rate was setto 0.3 mL/min, and the column effluent was monitoredcontinuously using UV detection at 368 nm. A C18 columnwas used and kept at a fixed temperature of 20°C ± 0.2. Theinjection volume was set to 5 μL.

Determination of the Pharmacokinetic Parameters

A non-compartmental analysis was performed usingWinNonlin® 1.5 software (Pharsight Co., Mountain View,CA, USA), and the following pharmacokinetic parameterswere calculated for the tested formulation and the prepareddrug suspension: Cmax (ng/mL), Tmax (h), AUC(0-264h) (ng.hr/mL), AUMC(0-t), and MRT (h).

Statistical Analysis of the Pharmacokinetic Parameters

Two-way ANOVA test followed by post hoc test wereperformed to determine the significance of difference be-tween the pharmacokinetic parameters (Cmax, AUC(0-t), andMRT) of the tested formulations as well as inter-subjectvariations using SPSS® software (IBM 20, USA). For Tmax-

initial, a non-parametric analysis of two independent variables(Mann-Whitney) was performed to compare the medians.

RESULTS AND DISCUSSION

In Vitro Drug Release Studies

The ISVs were prepared by emulsifying the NIS-TXmixture, dissolved in ethyl acetate, into an external oil phase(Captex®). Upon contact with the release medium (phos-phate buffer pH 7.4), the partially miscible solvent, ethylacetate, diffuses from the NIS solution into the aqueousenvironment, either directly or through the oil, leading to NISprecipitation and ISVs solidification (Fig. 1).

Safety was highly considered in selecting the materialsused in the formulation. NISs offer many advantages overother ionic surfactants; they are nontoxic, non-irritant, inaddition to being inert. They are commonly used in the

formulation of different oral and parenteral pharmaceuticalpreparations (21). According to the FDA guidelines, ethylacetate is included as an inactive ingredient for IM injections(40). Ethyl acetate has been used in the preparation of in situforming dosage forms (41–44) and has been proved to be lessmyotoxic than benzyl alcohol and triacetin with concentra-tions as large as 60% of the injected volume (undiluted) andequivalent to isotonic sodium chloride solution in itsmyotoxicity (45). Captex® 355 EP/NF is manufactured bythe esterification of caprylic/capric fatty acids and glycerin. Itis prepared from raw materials of vegetable sources.

The release profiles of TX from different ISVs formula-tions prepared are depicted in Fig. 2. All formulations showeda biphasic release pattern with an initial phase of rapid initialrelease phase followed by a delayed release phase thatextended for 216 h in some formulations.

High burst release observed in the formulations may beattributed in some formulations to the slow formation of thevesicles, owing to the slow diffusion of the solvent duringvesicle formation; whereas, in other formulations it may bedue to the extremely rapid diffusion of solvent that lead to thedecreased density of the obtained vesicles.

Statistical Analysis of the Experimental Design

Effect of Formulation Factors on Percentage Drug Releasedafter 1 h (Q1)

Table I shows the results of the percentage drug releasedafter 1 h (Q1). ANOVA test was performed to evaluate thelevel of significance of the tested factors on the measuredresponse. Results showed that only the type of NIS (A) had asignificant (p = 0.0006) effect on the percentage drug releasedafter 1 h (Q1). The change of the NIS type from Span® 60(HLB = 4.7) to Brij® 52 (HLB = 5.3) significantly increasedthe Q1 values. This could be due to that in the case of Span®60 based ISVs, particles with more hydrophobic matrix areformed; therefore, a slower release for the hydrophobic drugoccurs; consequently, the burst drug release for Span® 60based ISVs were lower than those for Brij® 52 based ISVs.Also, the two factor combinations, AC and BC, hadsignificant effects on Q1 values (p = 0.0480 and p = 0.0150,respectively). Figure 3a represents the 3D surface plots of theeffect of independent variables on Q1 values.

Effect of Formulation Factors on the MDT

Table I shows the values of the MDT. ANOVA test wasperformed to evaluate the level of significance of the testedfactors on the measured response, and showed that all of thethree independent factors had significant effects on the MDTvalues. Figure 3b and c represents the 3D surface plots of theeffect of independent variables on the MDT.

The type of NIS (A) had a significant (p < 0.0001) effecton MDT values. Replacing Brij® 52 with Span® 60 signifi-cantly increased the MDT values. This can be explained bythe fact that Span® 60 has a higher phase transitiontemperature (53°C) compared to that of Brij® 52 (32.5°C)which in turn decreases the fluidity and leakage of surfactantlayer, increasing the ability of the produced vesicles to retarddrug release (46).

1004 Ammar et al.

Results also revealed that the molar ratio of NIS toTween® 80 (B) had a significant positive effect on the MDTvalues. The increase in mole fraction of Tween® 80 led to asignificant increase in MDT values when using the phase ratio1:4 v/v. This could be attributed to the presence of an edgeactivator as Tween® 80 that decreased the interfacial tensionleading to better emulsification and a moderate solventdiffusion rate from the NIS droplets to the release medium.This led to the formation of dense vesicles and highentrapment of the drug.

On the other hand, the increase in molar ratio of Tween®80 to NIS from 1:8 to 1:4 lead to a decrease in the MDTvalues employing the phase ratio of 1:2 v/v. This may be dueto the fact that increasing Tween® 80 mole fraction beyond itsoptimum value increases bending of the chains decreasing thetightness of the bilayer leading to increased vesicle perme-ability (47). Further increase in the molar ratio from 1:4 to 1:2(Tween® 80 to NIS) employing the phase ratio of 1:2 v/vrevealed an increase in the MDT values.

Results indicated that the phase ratio (C) had asignificant positive effect on the MDT values. The increasein phase ratio from 1:4 to 1:2 v/v (internal to external phase)significantly increased the MDT values except for theformulations ST3 and BT2. The increase in phase ratio mighthave lowered the rate of solvent diffusion and deposition ofthe vesicles leading to the formation of denser matrix andhence retard the drug release. Furthermore, increasing theinternal phase ratio means increasing the amount of Span® 60or Brij® 52 and Tween®80 available to form the vesicles andthus higher ability to encapsulate the drug (moreNIS:Tween®80: drug ratio) which resulted in vesicles charac-terized by higher MDT values.

Effect of Formulation Factors on Mean PS Values of theObtained Vesicles

The average PS and size distribution (PDI) of theobtained vesicles were determined by dynamic light scattering(DLS) analysis using ZetaSizer. The ISVs obtained weremostly in the nano-range except for three formulations (ST5,ST6, and BT3). Table I represents the mean PS values of theobtained vesicles of the different prepared ISVs.

ANOVA test was performed to evaluate the level ofsignificance of the tested factors on the mean PS values of theobtained vesicles. Results showed that the type of NIS (A)

and the molar ratio of NIS:Tween®80 (B) had a significant(p = 0.0402 and p = 0.0008, respectively) effect on the meanPS of the obtained vesicles. Also, the two factor combinationsAB and BC had a significant (p = 0.0265 and p = 0.0008,respectively) effect on the PS values of the obtained vesicles.Figure 3d represents the 3D surface plots of the effect ofindependent variables on the PS of the obtained vesicles.

The type of NIS used (A) significantly affected the PSvalues of the obtained vesicles. Replacing Span® 60 withBrij® 52 significantly increased PS values in the phase ratio1:4 v/v; whereas, it significantly decreased PS values in thephase ratio 1:2 v/v. This can be attributed to the lower HLBof Span® 60 (HLB = 4.7) which increased the lipophilicity ofthe internal phase droplets in the lower phase ratio (1:4 v/v)leading to faster diffusion of the solvent into the release

Fig. 1. ST3 formulation before (a) and after (b) in vitro drug release studies with a magnified TEM picture

Fig. 2. Percentage tenoxicam released from in situ formed vesicles

1005Non-ionic Surfactant Based In Situ Forming Vesicles

Table I. Composition, Percentage Drug Released after 1 h (Q1)

Formulation code Composition Q1 (%) MDT (h) PS (nm) PDI

Type of NIS Molar ratio of NISto Tween® 80

Phase ratio (v/v) ethylacetate to Captex®

ST1 Span® 60 8:1 1:4 68.37 ± 2.32 2.18 ± 0.09 255.45 ± 39.36 0.492ST2 Span® 60 4:1 1:4 59.94 ± 3.05 14.12 ± 0.91 581.58 ± 238.75 0.83ST3 Span® 60 2:1 1:4 37.32 ± 3.56 21.56 ± 3.12 379.08 ± 69.15 0.643ST4 Span® 60 8:1 1:2 57.93 ± 0.13 18.97 ± 1.09 327.93 ± 9.47 0.271ST5 Span® 60 4:1 1:2 64.31 ± 2.55 15.21 ± 1.99 1301.18 ± 25.75 0.436ST6 Span® 60 2:1 1:2 65.70 ± 2.33 15.10 ± 1.08 1491.83 ± 288.07 0.358BT1 Brij® 52 8:1 1:4 80.21 ± 2.56 0.64 ± 0.03 365.37 ± 29.27 0.726BT2 Brij® 52 4:1 1:4 67.39 ± 1.54 3.16 ± 0.15 183.08 ± 33.02 0.504BT3 Brij® 52 2:1 1:4 74.78 ± 2.57 8.20 ± 1.22 1266.58 ± 212.93 0.387BT4 Brij® 52 8:1 1:2 65.72 ± 2.85 14.99 ± 1.03 280.56 ± 54.69 0.573BT5 Brij® 52 4:1 1:2 77.02 ± 0.21 1.83 ± 0.01 393.57 ± 19.67 0.723BT6 Brij® 52 2:1 1:2 64.67 ± 5.03 9.65 ± 0.73 505.18 ± 117.48 0.402

NIS non-ionic surfactant, MDT mean dissolution time, PS particle size, PDI polydispersity index for tenoxicam-loaded in situ forming vesicles

Fig. 3. 3D surface plot of the effect of concentration of type of NIS (A) and molar ratio ofNIS to Tween®80 (B) on a percentage drug released after 1 h (Q1), b and c meandissolution time (MDT), and d particle size (PS) for TX-loaded ISVs

1006 Ammar et al.

medium. This resulted in the formation of less dense vesicleswith larger size. On the other hand, this was balanced in thehigher phase ratio (1:2 v/v), leading to the prevalence of theclassical characteristics of Span® 60 based vesicular systems(smaller PS vesicles due to its longer alkyl chain) (48) and thelack of the large hydrophilic head of Brij® 52 that is highlyhydrated in aqueous medium forming larger PS values for theresulted vesicles.

The molar ratio of NIS:Tween® 80 (B) had asignificant effect on the PS of the obtained vesicles inthe phase ratio 1:2 v/v. The increase in Tween® 80 molefraction decreases the interfacial tension which results inincreasing the emulsification and the rate of diffusion ofthe solvent leading to the rapid deposition of less denselarge sized vesicles.

The PDI values for the prepared ISVs ranged from 0.27to 0.83 (Table I). Such variation in the values of PDI wouldsuggest the critical effect of ISV compositions and emulsionphase ratio on such parameter.

Assessment of In Vitro Drug Release Kinetics

Release profiles of all formulations were found to followthe Korsmeyer-Peppas diffusion model. All analyzed formu-lations were found to follow Fickian transport except for ST3(n 0.5 indicating non-Fickian anomalous transport). Thismay be attributed to the viscoelastic nature of obtainedvesicles which introduced relaxation time in the response(49).

Optimum target drug release pattern was observed inST3 compromising Span® 60 and Tween® 80 in the molarratio of 2:1 dissolved in ethyl acetate as the internal phase andemulsified into Captex® as the external oily phase, where itshowed an initial drug release of 37.32% in the first hourfollowed by a sustained drug release pattern for 6 days. Thus,it was chosen for further investigations.

Transmission Electron Microscopy

The determination of the morphological pattern of theoptimized formulation obtained vesicles using TEM showedthe formation of spherical nano-vesicles (Fig. 1). Theobtained vesicles exhibited a dense core with a less denseoutline. This may be elucidated with the deposition kinetics ofthe vesicles. After injection of the ISVs into the releasemedium, the solvent diffuses into the aqueous environmentand the release medium penetrates into the surface of theinternal phase droplets. The partially water miscible solventin the internal phase droplets had a slow diffusion rate uponcontact with the aqueous release medium which resulted inthe formation of dense vesicles. On the other hand, the lessdense outline may be due to the surface penetration of therelease medium to the vesicles during their deposition.

Differential Scanning Calorimetry

Figure 4 shows the DSC thermograms of Span® 60, TX,in addition to the optimized TX-loaded formulation, ST3.Span® 60 thermogram shows one main characteristic endo-thermic peak at 53°C, which corresponds to Span® 60transition temperatures. TX thermogram shows one sharp

endothermic peak at 223.89°C which indicates the presence ofthe crystalline form of the drug and represents its meltingpoint. This endothermic peak ends with an exothermic peakat 226.5°C, which may be due to the decomposition of thedrug when reaching its melting point (50). The optimizedformulation (ST3) thermogram shows a peak at 56.66°C anddisappearance of the peak for TX. The slight shift in the peakof Span® 60 from 53 to 56.66°C with more broadnessindicates that Span® 60 preserved its structure while formingan ordered structure of vesicles with other excipients. Thedisappearance of the drug peak confirms the solubilization ofthe amorphous form of the drug and its incorporation insidethese vesicles (51,52).

Viscosity Measurements

As computed from Farrow’s equation, Farrow’s constantwas found to be 0.9798 which is approximately unity. Thisindicates a nearly ideal Newtonian behavior of the examinedformulation where the viscosity is almost constant uponincrease in shear stress applied (ɳ at maximum shearstress = 10.99 cP and ɳ at minimum shear stress = 10.46 cP).

Injectability

The injectability of the investigated formulations wasassessed by comparing their mean flow rate (39). Byanalyzing the results for the investigated formulations, it canbe observed that under constant pressure, the marketed oilyinjection (Betolvex™) took longer time to pass through a 19-gauge needle. The flow rate for Betolvex™ injection wasrecorded to be 0.97 mL/min. On the contrary, the selectedformulation, ST3, succeeded to flow rapidly when comparedto the market product. The mean flow rate taken for ST3injection was 5.45 mL/min. This higher flow rate indicatesgood injectability for the prepared formulation.

In Vivo Evaluation of Selected TX ISVs

In our study, in vivo evaluation of the selected TX-loaded ISV was carried out by measuring the plasmaconcentration of the drug over a period of 264 h after IMinjection of 1.5 mL of either the selected ISV or a drugsuspension in phosphate buffer (pH 7.4) (both equivalent to1 mg/mL of the drug). No local injection-site reactions(redness, itching, or swelling) were recorded for the testedformulations on the following days after the IM injections.

The selected formulation (ST3) prepared using Span® 60and Tween® 80 (molar ratio of 2:1) dissolved in ethyl acetateas the internal phase and Captex® as the external phase in theratio of 1:4 v/v (internal phase to external phase) was chosenafter extensive in vitro investigation, to perform the in vivostudies. ST3 had the required release profile as it achieved amoderate initial drug release (37.32%) in the first hourfollowed by sustained drug release over 144 h. A cross-overdesign was adopted in the in vivo study. All the rabbits fullycompleted the study. No adverse reactions were reportedduring the study.

The mean plasma concentrations versus time for theinvestigated formulation (ST3) and the drug suspension aregraphically represented in Fig. 5. In both tested formulations,

1007Non-ionic Surfactant Based In Situ Forming Vesicles

TX was detected in plasma as fast as 30 min following the IMinjection. The level of TX in plasma of rabbits receiving thedrug suspension remained detectable until 24 h, with themaximum level of the drug achieved after 0.5 h. On the otherhand, the plasma drug concentration following IM injectionof the optimized ISV formulations remained detectable until264 h, with the maximum level achieved after 96 h. Suchbiphasic pattern with an initial fast drug release followed by asustained drug release that extended for 11 days is consistentwith the obtained results from the in vitro release study. Forthe Tmax, only the values for the initial phase (Tmax-initial)were compared to that for the suspension as it has only onedrug release phase.

No significant difference (p > 0.05) was observedbe tween Cma x and Tma x - i n i t i a l v a l u e s o f ST3(632.35 ± 158.28 ng/mL and 1.00 ± 0.61 h, respectively) andthose of the drug suspension (470.22 ± 138.31 ng/mL and 0.50 ± 0.49 h, respectively), ensuring that the invented ISVs(ST3) could readily deliver the drug in approximately thesame time and extent as the suspension.

Determination of the area under the curve (AUC) isconsidered an important parameter for identifying theavailability of the drug from the tested formulations. Asignificant difference (p = 0.0012) was observed betweenAUC values of the optimized formulation (ST3) and that of

the drug suspension (85,317.00 ± 31,303.16 and1818.23 ± 668.25 ng.hr/mL, respectively), where ST3 exhib-ited a 45-fold higher AUC value compared to that of the drugsuspension, indicating much better availability of the drugfrom the ISVs compared to the drug suspension. This isobviously attributed to the deposition of the ISV vesiclesupon contact with the aqueous medium at the injection siteand the incorporation of the drug inside its core. Thiscontrolled the drug release, highly prolonged its action, andmaximized its availability inside the body compared to thefree drug in the injected suspension. In addition, the smallsize of the obtained nano-sized vesicles prevented theirclearance and thus they remained in the body acting as adepot for a long time (53,54).

Finally, the duration of action for the tested formulationswas determined by their MRT values. Statistical analysisshowed a significant difference (p < 0.0001) between ST3 andthe drug suspension MRT values. The extreme increase in theMRT value from 3.48 ± 1.65 h for the injected drugsuspension to 98.52 ± 9.67 h for the injected ST3 confirmedthat the candidate formulation (ST3) was highly efficient tosustain the drug release. Upon contact of the ISVs with theaqueous medium at the injection site, the internal phasesolvent diffuses into the aqueous medium with the depositionof dense vesicles incorporating the drug and controlling itsrelease. These vesicles act as depot systems for the drugprolonging its action inside the body.

The above mentioned data would, definitely, point outthe potentiality of formulating TX in the form of ISVs usingNIS as a substitute for polymers used for the formulation of insitu forming microparticles with the use of self-emulsifyingoils as Captex® that could successfully achieve an acceptablyrapid initial drug release that provides a suitable onset ofaction as well as a sustained drug release reducing dosingfrequency.

CONCLUSION

In this study, 22.31 full factorial experimental design wasadopted for the preparation of TX-loaded, NIS based, in situforming vesicles. Results revealed that only the type of theNIS had a significant (p = 0.0006) effect on cumulative

Fig. 4. DSC thermograms of (a) Span® 60, (b) tenoxicam, and (c) tenoxicam-loaded ST3

Fig. 5. Mean plasma concentration of tenoxicam following IMadministration of ST3 and drug suspension

1008 Ammar et al.

percentage of drug released after 1 h. Whereas, both thetype of NIS and the molar ratio of NIS: cholesterol had asignificant (p = 0.0402 and p = 0.0008, respectively) effecton the mean PS of the obtained vesicles. The invented insitu forming vesicles succeeded to improve thepharmacokinetic parameters of TX compared to the drugsuspension when administrated IM where it extremelyprolonged its duration of action and increased itsbioavailability. The abovementioned data would,definitely, point out the potential of formulating TX inthe form of in situ forming vesicles using non-ionicsurfactants as a substitute for expensive polymers usedfor the formulation of the classical in situ formingmicroparticles. All of which could successfully achieve anacceptably rapid initial drug release that provides asuitable onset of action as well as a sustained drug releaseover more than 10 days reducing dosing frequency.

COMPLIANCE WITH ETHICAL STANDARDS

All animal procedures were performed according to theprotocols reviewed and approved by the Research EthicsCommittee of Faculty of Pharmacy, Cairo University.

Conflict of Interest The authors declare that they have noconflicts of interest.

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