Contents lists available at ScienceDirect

Colloids and Surfaces A

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

Influence of crosslinked alginate on drug release from highly concentratedemulsions

Pablo Bonillaa, Eva M. Ariasa, Conxita Solansb,c, María José García-Celmaa,c,⁎

a University of Barcelona, Department of Pharmacy and Pharmaceutical Technology and Physicochemistry, Institute of Nanoscience and Nanotechnology (IN2UB), Avda,Joan XXIII s/n 08028 Barcelona, Spainb Spanish Council for Scientific Research (CSIC), Institute of Advanced Chemistry of Catalonia (IQAC), C/Jordi Girona 18-26, 08034 Barcelona, Spainc Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain

G R A P H I C A L A B S T R A C T

A R T I C L E I N F O

Keywords:High internal phase ratio emulsionDiffusionControlled drug releaseCrosslinked calcium alginateKetoprofenClindamycine hydrochloride

A B S T R A C T

Biocompatible water-in-oil (W/O) and oil-in-water (W/O) highly concentrated emulsions (HIPREs) have beenformed at 25 °C, in systems consisting of aqueous component/castor oil derivative surfactant/oil compound. Inboth kinds of emulsions the concentration of internal phase was 83% and the aqueous component consisted ofwater, 1% sodium alginate solution or a mixture of sodium alginate solution and calcium chloride solution(crosslinked calcium alginate). Ketoprofen (KP) or clindamycine hydrochloride (CH) were solubilized in bothkinds of HIPREs and the influence of the composition of the aqueous phase on drug release was studied. Dropletsizes of the W/O HIPREs formed were in the nanometric range, being smaller than other HIPREs described in theliterature. Dynamic viscoelastic measurements showed an increase in the elastic modulus (G’) of the emulsions inthe presence of alginate or crosslinked alginate. The influence of the order of incorporation of alginate andcalcium chloride on the viscosity was demonstrated, as an indication of crosslinking efficiency. KP release to aPBS receptor solution was slower when crosslinked alginate was formed in the internal phase of W/O HIPREs. Incontrast, the influence of crosslinked alginate on the release of CH was negligible and the solubility of the drug inthe disperse phase demonstrated to be the main mechanism responsible of CH diffusion. The results obtainedsuggest that the formation of a crosslinked alginate matrix in the internal phase of HIPREs is a factor that has tobe taken into account in the development of controlled drug delivery systems.

1. Introduction

Highly concentrated emulsions, also known as high internal phaseratio emulsions (HIPRE) are emulsions in which the volume fraction ofthe dispersed phase is higher than 0.74, the maximum volume fraction

of close-packed mono-disperse spheres [1]. The droplets of HIPREs aredeformed showing polyhedral shapes and are separated from each otherby a thin film of continuous phase, which make them microscopicallysimilar to foams [2,3] As a consequence of their structural features, therheological properties of these emulsions may range from elastic,

http://dx.doi.org/10.1016/j.colsurfa.2017.07.026Received 30 September 2016; Received in revised form 5 April 2017; Accepted 8 July 2017

⁎ Corresponding author at: University of Barcelona, Faculty of Pharmacy and Food Sciences, Department of Pharmacy and Pharmaceutical Technology and Physicochemistry. Instituteof Nanoscience and Nanotechnology (IN2UB), Avda. Joan XXIII s/n, 08028 Barcelona, Spain.

E-mail address: mjgarcia@ub.edu (M.J. García-Celma).

Colloids and Surfaces A 536 (2018) 148–155

Available online 11 July 20170927-7757/ © 2017 Elsevier B.V. All rights reserved.

T

typical of solid materials, to viscoelastic [2]. As ordinary emulsions,HIPREs can be classified into two types, water-in-oil (W/O) and oil-in-water (O/W) according to the polarity of the internal and the externalphases. Owing to their interesting properties, HIPREs are being used inmany applications [1]. In the pharmaceutical field, their potential ascontrolled drug delivery systems has already been evidenced in pre-vious studies [4–10], but they can also be used as reaction media forenvironmentally friendly molecular synthesis [11,12] and preparationof low density organic and inorganic materials with potential applica-tions as scaffolds in tissue engineering [13–15].

In general, it is observed that the release of active substances fromemulsions and particularly from HIPREs is slower than from solutionswhen contained in a dialysis bag [4,16]. Systematic studies on the re-lease of molecules from HIPREs confirmed the dependence of the re-lease on the partition coefficient of the diffusing molecule but also onthe properties of the interfacial film. Other factors have been reported,such as the surfactant chain length [4], surfactant mixtures [5,6], oiltype [5], oil/surfactant ratio [6], dispersed phase fraction and the ionicstrength of the aqueous disperse phase [4,17], that allow to control therelease of molecules through their influence on the stability of theHIPREs (interfacial film cohesion) [18], and on the partition coefficientof the diffusing molecules [4,17,18]. These results provided evidencethat the interface is a more effective barrier to the diffusion of mole-cules than the external phase of the HIPRE [4,19]. Furthermore, dropletsize and oil film thickness have also been proposed to influence thetransport rate of molecules in W/O HIPREs [20]. Based on the dataavailable, our group proposed a mechanism of release from HIPREsconsisting of two simultaneous stages: the passage of the diffusingmolecule through the interfacial film of the dispersed phase drops(which is the limiting stage) and the diffusion in the continuous phase[5,7]. Although much progress has been done in the last two decades onthe understanding and control of the release of molecules from HIPREs,there are still aspects which remain unclear. A challenging point con-cerns the effect of the nature of the diffusing molecule on its releasefrom HIPREs. Several features of the diffusing molecule such as mole-cular weight, interfacial properties, and solubility, have been claimed toplay a role on the release from W/O HIPREs, but their specific effect hasnot yet been completely clarified.

The incorporation of polymers in the continuous or in the dispersedphase of HIPREs may confer them specific properties. These emulsionscan be used as templates for producing either particles or solid foamswith very high pore volume, when selecting the internal or the externalphase for polymer deposition, followed by the removal of the otherphase components [21–23]. Alginate is a natural anionic poly-saccharide that consists of linear chains of α-L-guluronic acid and β-D-mannuronic acid residues joined by 1, 4-glycosidic linkages. Alginatehas been used in drug delivery systems due to its biocompatibility,water solubility, sol-gel transition properties, mucoadhesion, biode-gradability, film formation properties, low cost and availability [24].Divalent cations, such as calcium ions, interact with the negativelycharged carboxyl groups from the guluronic blocks of alginate to formthe so-called “egg-box” structure [25,26] (Fig. 1). Soluble sodium al-ginate can be physically crosslinked using calcium chloride [27] bymixing the two solutions, what is a simple process useful for pharma-ceutical applications. In the so-called diffusion method or external ge-lation method, the alginate solution is delivered to the calcium chloridesolution and gel formation occurs as crosslinking cations diffuse intothe alginate solution [24]. The emulsification of alginate solutions indifferent oil phases followed by gelation of the alginate droplets inconventional W/O emulsions to form alginate particles has been re-ported in the literature [27]. The influence of crosslinked polymers ondrug release from emulsions is a challenging point that has not beensystematically investigated. The aim of the present research was tostudy the influence of crosslinked calcium alginate in the internal andexternal phase of HIPREs on the release properties of W/O and O/WHIPREs in order to develop controlled drug delivery systems and

templates for the preparation of nanostructured drug delivery systems,both particulated materials and solid foams. Most of the studies on therelease of actives from HIPREs have been performed with W/O emul-sions. In the present work, to better understand the influence of alginateon the release behavior, both kinds of HIPREs, W/O and O/W havebeen investigated. The release of two model drugs with differentcharacteristics: a lipophilic drug, ketoprofen (KP) and a hydrophilicdrug, clindamycin hydrochloride (CH), from both type of HIPREs hasbeen analyzed as a function of composition variables, such as sodiumalginate and calcium chloride concentration, and methodology ofemulsion preparation.

2. Material and methods

2.1. Materials

Sodium alginate of low viscosity (250cPs) from brown algae wasobtained in solid state from Sigma-Aldrich, calcium chloride solution1 M was also obtained from Sigma-Aldrich. Cremophor WO7 (PEG-7Hydrogenated Castor Oil) and Cremophor RH455 (PEG-40Hydrogenated Castor Oil, 90% in water/propylene glycol) from BASFwere used as surfactants. Liquid paraffin was purchased from Merck.Miglyol 812®, a mixture of neutral esters of saturated coconut andpalmkernel oil-derived caprylic and capric fatty acids and glycerin orpropylene glycol with viscosity 27–33 cPs at 20 °C, was obtained fromFagron Ibérica S.A.V. Phosphate buffer solution pH 7.4 (PBS) was usedas receptor solution in the release studies. It was prepared with KH2PO4

from Fagron Iberica S.A.V., Na2HPO4 from Probus S.A., and NaCl fromAcofarma. Water was deionized by MilliQ®

filtration. Ketoprofen (KP)and Clindamycin hydrochloride (CH) (Table 1) both with 99.8% puritywere purchased from Fagron Iberica. The hydrophilic membrane used inrelease studies was a cellulose tubular membrane Cellu SepT3® with anominal molecular weight cut-off (MWCO) of 12000–14000 Da, fromOrange Scientific. Commercial gel used as a reference was Fastum Gel™(Guidotti Farma) which contains 2.5% of ketoprofen and the excipientsare carbomer, ethanol (96%), diethanolamine, methyl para-hydroxybenzoate, propylparahydroxybenzoate, essence, and water.

2.2. Methods

2.2.1. Formation of alginate-calcium colloidal dispersionsSodium alginate solutions were prepared by adding sodium alginate

to water at 25 °C; then, the samples were thoroughly stirring with ahigh speed homogenizer (Ultraturrax ™ T25) at 8000 rpm. Various so-dium alginate solutions were mixing with calcium chloride solutions atdifferent concentrations to obtain colloidal solutions and gels.

2.2.2. Formation of highly concentrated emulsionsW/O and O/W HIPREs were prepared by the method of successive

additions [8]. These emulsions were obtained by stepwise addition ofthe internal phase to the external phase at 25 °C while stirring at2600 rpm. HIPRE formulations were prepared using as aqueous com-ponent: a) water, b) an alginate solution (1 wt.%), c) a calcium chloridesolution (0.015 wt.%) or d) a mixture of alginate sodium (1 wt.%) andcalcium chloride (0.015 wt.%). Additionally some W/O and O/WHIPREs were prepared by dissolving ketoprofen in a mixture of oil andsurfactant or clindamicyne hydrochloride in the aqueous component,prior to the emulsion formation. The composition of W/O and O/WHIPREs are shown in Table 2 and 3. The influence of the order of in-corporation of alginate sodium and calcium chloride solutions onHIPREs stability and drug release was determined.

A model lipophilic drug (KP) or a model hydrophilic drug (CH) wereincorporated in both W/O and O/W HIPREs. The same amount of eachdrug was solubilized in the emulsions for comparative purposes; ac-cordingly, the concentration was 0.25% of KP or CH in W/O HIPREs(i.e. 2.5 mg/g of HIPRE) and 1.5% of KP or CH in O/W HIPREs (i.e.

P. Bonilla et al. Colloids and Surfaces A 536 (2018) 148–155

149

15 mg/g of HIPRE). The amount of active to be incorporated in theHIPREs was chosen taking into account the maximum solubility of thedrugs in the dispersed and continuous phases of the emulsions, thetherapeutic dose and also the accomplishment of sink conditions in thereceptor solution during the release experiments.

2.2.3. Emulsions characterization and stability assessmentQualitative estimation of droplet size evolution as well as detection

of crystallization phenomena were assessed by optical microscopy, witha Leica DMIL LED equipment. W/O HIPREs were also characterized byscanning electronic microscopy (SEM), with a JEOL JEM-1010 equip-ment. The stability of the emulsions was determined by visual ob-servation of appearance of phase separation and/or fluency at 25 °C.

2.2.4. Rheological characterizationThe rheological properties of HIPREs were studied by determining

the elastic modulus (G’) and the viscous modulus (G”) as a function offrequency. Dynamic viscoelastic measurements were performed at25 °C by using a rheometer AR-G2®RS1 (TA Instruments, Ltd.). Themeasurements were performed within the linear viscoelastic region. Nowall slip was observed. The geometries used were standard steel par-allel plates of 40 mm (smooth geometries).

2.2.5. Drug solubility determinationThe solubility of KP or CH in different solvents was determined in

triplicate by adding an excess of the drug to a small amount of solvent,at 25 °C. Each sample was thoroughly stirred and the centrifuged at25 °C at 3500 rpm for 5 min. Afterwards, it was allowed to stabilizeovernight in a water bath at 25 °C. The supernatant was analyzed byHPLC.

2.2.6. Release experimentsIn vitro release studies were carried out by the dialysis bag method

[10]. Approximately 1 g of HIPRE containing ketoprofen or clin-damicyne hydrochloride was placed in a dialysis bag and submerged in150 mL of receptor solution (PBS, phosphate buffer solutionpH = 7.4).The dialysis bags were porous hydrophilic membranes witha molecular weight cut off (MWCO) much bigger than the molecularweight of ketoprofen or clindamycine. The diffusion cells consisted ofcylindrical thermo jacket glass vessels connected to a water bath set at25 °C and closed to avoid loss of receptor solution by evaporation. Thereceptor solution was stirred by means of a magnetic stirrer. In order todetermine the amount of drug released as a function of time, 1 mL ofreceptor solution was removed for analysis and the same amount of PBSsolution was replaced. The release study lasted for 24 h. Aliquots ofreceptor solution were withdrawn for determination of drug con-centration by HPLC. At least four replicates have been studied in eachcase. The experimental error has been calculated as the standard de-viation.

2.2.7. Determination of KP and CH concentration by HPLCKP and CH were analyzed by Shimadzu HPLC equipment. For KP

determination, a Kromasil® 100-5C18 column was used and the UVdetection was set at 233 nm. Separation was carried out at room tem-perature using 55% acetonitrile and 45% aqueous phase (pH 3.0), asthe mobile phase, with a flow rate of 1 mL/min and an injection volumeof 20 μL. The KP retention time was approximately 7 min. For CH de-termination, a 5 μm × 15 cm× 0.46 cm Spherisorb®ODS column wasused and the UV detection was set at 210 nm. Separation was carriedout at room temperature using acetonitrile—solution of potassium di-hydrogen phosphate adjusted to pH 7.5 with a solution of potassiumhydroxide (8 N) (450:550, v/v) as the mobile phase, with a flow rate of1 mL/min, and an injection volume of 100 μL. The CH retention time

Fig. 1. Crosslinking of alginate by calcium cations, resulting in “egg-box” calcium junctions [26].

Table 1Chemical structure and properties of Ketoprofen (KP) and Clindamycin hydrochloride (CH).

Ketoprofen (KP) Clindamycin hydrochloride (CH)

MW: 254.29 MW: 461.44pKa: 4.45 pKa: 7.6log P: 0.97 log P: 2.16

P. Bonilla et al. Colloids and Surfaces A 536 (2018) 148–155

150

was 6 min.

3. Results and discussion

3.1. HIPREs preparation and characterization

Crosslinked calcium alginate colloidal solutions were prepared bymixing at once sodium alginate and calcium chloride solutions atconstant ratios at 25 °C. Viscosity determinations of these mixtures justafter preparation were assessed in order to select compositions suitableto be the aqueous component of HIPREs. Alginate concentrations higherthan 2 wt% or calcium chloride higher than 0.05 wt% showed highviscosity values (Fig. 2). The mixture composed of sodium alginate(1 wt%) and calcium chloride (0.015 wt%) presented low viscosity at25 °C (19.6 ± 0.2 mPa s) and was considered the optimal for HIPREsformation. The viscosity of the mixture selected showed no significantdifferences when the order of incorporation was changed and gelifica-tion was not observed.

W/O and O/W HIPREs with 83% of dispersed phase were prepared.Formation of W/O-HIPREs in the water/Cremophor WO7/liquid par-affin system at 25 °C was investigated previously [8]. W/O HIPREsprepared in this work consisted of aqueous component/CremophorWO7/liquid paraffin, with an oil/surfactant weight ratio of 70/30,while O/W HIPREs consisted of aqueous component/CremophorRH455/Miglyol 812, with an aqueous component/surfactant weightratio of 80/20. Aqueous phase composition is described in 2.2.2 Sec-tion. The influence of the composition of the aqueous phase on HIPREsstability was studied by visual observation of appearance of phase se-paration and/or fluency at 25 °C, microscopy characterization andrheology measurements. None of the HIPREs prepared showed phaseseparation after 3 months. However, the W/O HIPREs with only

calcium chloride solution as aqueous phase became more fluent afterseveral hours of preparation and were discarded for further studies.Fig. 3 shows microscopy images of W/O and O/W HIPREs preparedwith water or crosslinked sodium alginate as aqueous components.

It is noteworthy that the W/O HIPREs presented smaller dropletsizes compared to O/W HIPREs (15–40 μm). This fact could be attrib-uted to the higher amount of surfactant, 5.1 wt%, in W/O HIPREs, andonly 3.4 wt% in the O/W emulsions. The microscopic aspect of O/WHIPREs (Fig. 3c and d) reveals a high density of polydisperse dropletswith a predominance of droplets of the small size (lower than 20 μm)over those showing large sizes (above 30 μm).The nanometric size ofW/O emulsions could be characterized by electron microscopy, asshown in Fig. 4. Droplet sizes of W/O HIPREs ranged from 60 to600 nm, being smaller than other HIPREs described in the literature. A

Table 2Composition of W/O HIPREs.

W/O HIPRE O/Sa weight ratio Aqueous component Drug Aqueous component: Order of incorporation

Water, wt% Sodium alginate, wt% CaCl2, wt% KP, wt% CH, wt%

HWO 70/30 83 waterHWOA 1 alginateHWOAC 1 0.015 1st alginate, 2nd CaCl2HWOCA 1 0.015 1st CaCl2, 2nd alginateHWOac 1 0.015 alginate-Ca solution previously preparedHWOKP 0.25 waterHWOAKP 1 0.25 alginateHWOACKP 1 0.015 1st alginate, 2nd CaCl2HWOCAKP 1 0.015 1st CaCl2, 2nd alginateHWOacKP 1 0.015 0.25 alginate-Ca solution previously preparedHWOCH 0.25 waterHWOACH 1 0.25 alginateHWOacCH 1 0.015 0.25 alginate-Ca solution previously prepared

a O/S: oil/surfactant.

Table 3Composition of O/W HIPREs.

O/W HIPRE W/Sa weight ratio Aqueous component Drug Aqueous component: Order of incorporation

Water, wt% Sodium alginate, wt% CaCl2, wt% KP, wt% CH, wt%

HOW 80/20 13.6 waterHOWac 1 0.015 alginate-Ca solution previously preparedHOWKP 1.5 waterHWOAKP 1 1.5 alginateHOWacKP 1 0.015 1.5 alginate-Ca solution previously preparedHOWCH 1.5 waterHOWACH 1 1.5 alginateHOWacCH 1 0.015 1.5 alginate-Ca solution previously prepared

a W/S: water/surfactant.

0

50

100

150

200

250

300

350

400

0 0.02 0.04 0.06 0.08 0.1

Visc

osity

(mPa

· s)

CaCl2 (%)

Alginate 2%Alginate 1%Alginate 0,75%Alginate 0,5%Alginate 0,25%Alginate 0,06%

Fig. 2. Viscosity of various sodium alginate and calcium chloride mixtures as a functionof calcium chloride concentration, just after preparation at 25 °C.

P. Bonilla et al. Colloids and Surfaces A 536 (2018) 148–155

151

significant influence of alginate, calcium chloride or crosslinked algi-nate on the emulsions droplet sizes could not be clearly observed in theHIPREs.

Studies on the rheological properties of W/O HIPREs formed in si-milar systems showed [28,29] that they have a viscoelastic responsethat can be fitted to a Maxwell model: an elastic modulus produced byinterfacial area increase and a viscous modulus produced by the losscaused by slippage of droplets against droplets. Oscillatory measure-ments performed in W/O HIPRE of the water/Cremophor WO7/liquidparaffin system, with an oil/surfactant weight ratio of 60:40 and 90%water content, reported in the literature [8], showed a slight depen-dence of the storage modulus (G’) with frequency at low values, but itreached a constant value of 5 × 102 Pa at frequencies higher than

10 Hz. In the present work, the rheological properties of W/O and O/WHIPREs as a function of aqueous phase composition were evaluated.Fig. 5 shows the influence of the aqueous composition of W/O HIPREsand the order of incorporation of sodium alginate and calcium chlorideon the elastic modulus (G’) and the viscous modulus (G”) values.

As shown in Fig. 5, in all the W/O HIPREs studied, the elasticmodulus (G’) is higher than the viscous modulus (G”). The G” value isaround 300 Pa for all the HIPREs, independently of the aqueous com-ponent. However, significant differences can be observed in the G’modulus, with the lowest values for the W/O HIPRE with only waterand the highest for the HIPRE with only alginate. The order of in-corporation of calcium chloride and sodium alginate influences con-siderably the value of the elastic modulus (G’), with higher values whensodium alginate is added after calcium chloride. A higher crosslinkingdegree should be related to a higher value of the elastic modulus, asdescribed previously in the literature with other polymers [30]. Thedifferences found in the present study could be attributed to diffusion ofcalcium ions into the alginate matrix. A more efficient crosslinkingcould be obtained in a highly compartimentalized media such as a W/OHIPRE, if calcium ions are present when sodium alginate is added. Thelower G’ values obtained in crosslinked alginate HIPREs with respect toalginate HIPREs could be attributed to the instability produced bycalcium chloride solution, that was previously observed in the HIPREswith only calcium chloride solution as aqueous component that showedfluency after several hours of preparation. The rheological behaviorobserved suggests that the presence of alginate or crosslinked alginatecould improve gel strength.

Rheology determinations were also performed with O/W HIPREsbut significant differences could not be observed probably due to thelow concentration of the aqueous component (only 13.6% of theemulsion) compared with the high amount in W/O HIPREs (83%).Then, the effect of alginate or crosslinked alginate could not be clearlyevidenced in O/W HIPREs.

No significant differences on droplet sizes or rheology properties

Fig. 3. Optical micrographs of W/O and O/WHIPREs with different aqueous components: a) W/OHIPRE with water, b) W/O HIPRE with crosslinkedsodium alginate, c) O/W HIPRE with water and d)O/W HIPRE with crosslinked sodium alginate.

Fig. 4. Electron microscopy (SEM) image of a W/O HIPRE containing crosslinked alginateas aqueous component.

P. Bonilla et al. Colloids and Surfaces A 536 (2018) 148–155

152

were observed with KP or CH incorporation to HIPREs. Visual inspec-tion of the emulsions confirmed no phase separation or other macro-scopic change after a storage period of 3 months.

3.2. Drug release studies

The lipophilic drug KP was solubilized in W/O HIPREs (0.25 wt%)and in O/W HIPREs (1.5%). The solubility of KP in the receptor solution(PBS at pH = 7.4) is 0.4 wt%. Prior to the experiments, it was calcu-lated that even if the total of ketoprofen present in any HIPRE wascompletely released, still in that case, and considering the volume of therelease media, the KP dissolved would be below the 20% of its max-imum solubility in PBS. Therefore, sink conditions were ensured inthese experiments and drug diffusion is not limited by the low solubilityin the receptor solution. Although the results obtained with the dialysisbag method are not comparable to those resulting in physiologicalconditions, the setup is useful for comparative studies between for-mulations.

Fig. 6 shows the profiles of KP released to a PBS receptor solution asa function of HIPRE composition. KP concentration in the W/O HIPREswas only 0.25% due to its low water and oil solubility. The aqueouscomponents of the W/O HIPREs studied were either water, or 1% so-dium alginate solution or a mixture of sodium alginate solution andcalcium chloride solution (crosslinked calcium alginate). A KP (2.5%)commercial hydrogel (Fastum gel™) and a KP (0.36%) PBS solutionwere also assayed for comparative purposes. Solutions are used in drugrelease experiments as a reference to evaluate the possible retention ofthe free-drug in the dialysis membrane.

KP release from W/O HIPREs is slower than the release from a PBS

solution and commercial hydrogel. These results are in agreement withthose reported in the literature based on different actives [10] anddosage forms [30]. The partition coefficient of the diffusing moleculebetween the continuous and the dispersed phase and the properties ofthe interfacial film have been proposed as the key factors for moleculardiffusion from HIPREs [9]. Taking into account the solubility of KP inthe components of the W/O HIPRE, it could be assumed that the activeis located preferently in the interphase, since it is practically insolublein water (0.001 wt%), very slightly soluble in liquid paraffin (0.04 wt%) but the solubility in the mixture liquid paraffin/Cremophor WO7(70:30) is 1.8 wt%. KP solubility in sodium alginate solution is 0.07 wt% and in the mixture sodium alginate/CaCl2 is 0.05 wt%, slightlyhigher than in water, then, the partition of the active could be modifiedas a function of the aqueous phase composition. However, it is note-worthy the slower release obtained from W/O HIPREs with crosslinkedalginate in the internal phase, with respect to the emulsion with onlywater as aqueous phase, especially after the first hours. This fact couldbe interpreted as the formation of a crosslinked alginate matrix in theinternal phase of HIPREs could retain KP molecules more than a non-structured internal phase. Taking into consideration the rheologicalbehavior reported above, it should be noted that viscosity of W/OHIPREs was lower for HIPREs with only water as internal phase, but thehighest with alginate solution as aqueous phase.

For a better understanding of the release behavior, KP release fromW/O HIPREs with different order of incorporation of sodium alginateand calcium chloride was studied. Fig. 7 shows the profiles obtainedwhen sodium alginate solution was added previously to calciumchloride solution and vice versa.

The release pattern shows significant differences related to the orderof incorporation of sodium alginate and calcium chloride. These resultssuggest that the slower KP release obtained when calcium chloridesolution is incorporated before sodium alginate, could be related to ahigher crosslinking efficiency and, then, the crosslinked alginate matrixformed in the W/O HIPREs internal phase could be responsible of theslower release of KP in comparison to W/O HIPREs without alginate.The small molecular size of KP could facilitate the retention into thecrosslinked alginate matrix formed in the emulsion. The properties ofthe disperse phase of a highly concentrated emulsion may influence therelease behavior of a molecule by modifying the partition between thedisperse and the continuous phase. The increased viscosity produced byalginate and the crosslinked matrix formed in the highly concentratedemulsion could be also responsible of the slower release observed witha lipophilic drug mainly solubilized in the interphase.

KP release from O/W HIPREs was also determined. Fig. 8 shows theprofiles of KP released to a PBS receptor solution as a function of HIPREcomposition. KP concentration in the O/W HIPREs was 1.5% due to thehigher solubility of the active in the oil component (Miglyol). This

0

200

400

600

800

0 2 4 6 8 10

HIPRE alginate (HWOA)HIPRE 1st alginate (HWOAC)HIPRE 1st calcium (HWOCA)

0

500

1000

1500

2000

2500

3000

3500

10G

` (P

a)

HIPRE alginate (HWOA)HIPRE 1st alginate (HWOAC)HIPRE 1st calcium (HWOCA)

Fig. 5. Viscoelasticity of W/O HIPREs as a functionof aqueous component composition and order of in-corporation of alginate solution and calcium chloridesolution in the HIPRE preparation. The elastic mod-ulus (G’) and the viscous modulus (G”) are plotted asa function of frequency. Data points are the mean offour measurements.

0

20

40

60

80

100

0 50 100 150 200 250 300 350 400 450

0.36% KP in PBSFastumGelHIPRE HWOKPHIPRE HWOAKPHIPRE HWOacKP

Fig. 6. Release profiles of KP from W/O HIPREs with different aqueous componentcompositions, from a PBS solution and from a commercial gel.

P. Bonilla et al. Colloids and Surfaces A 536 (2018) 148–155

153

amount allowed sink conditions accomplishment in PBS receptor solu-tion. The aqueous components of the O/W HIPREs studied were eitherwater or a mixture of sodium alginate solution and calcium chloridesolution (crosslinked calcium alginate), with a final concentration of1% sodium alginate and 0.015% calcium chloride. A KP (2.5%) com-mercial hydrogel and a KP (0.36%) PBS solution and a KP (1.5%) inMiglyol 812 were also assayed for comparative purposes.

The results obtained show a faster diffusion from solutions (PBSsolution and Miglyol solution) and commercial gel (Fastum gel) to thereceptor solution in comparison to O/W HIPREs. After 2 h, more than50% of KP from these solutions and commercial gel could be found inthe receptor solution. The slower release in the first hours from the oilsolution could be attributed to the hydrophilic nature of the membrane.The retention of KP in O/W HIPREs could be explained by the highersolubility of the active in the internal phase (4 wt% in Miglyol). Thiseffect has been described before in the literature [9]. No significantdifferences were observed in KP release from O/W HIPREs as a functionof the continuous phase composition, specially during the first 3 h. Thedifferent behavior observed in O/W and W/O HIPREs with crosslinkedalginate described before can be related to the low total concentrationof alginate in the aqueous phase of both kinds of emulsions: while theaqueous phase is 83% of the W/O HIPRE, it is only 13.6% in the O/WHIPRE and then, the total amount of alginate is considerably differentin both kinds of emulsions (0.83% in W/O HIPREs and 0.136% in O/WHIPREs), despite the ratio between sodium alginate and calciumchloride is constant. The influence on KP release of the structure formedby crosslinked alginate in the continuous phase of O/W HIPREs isnegligible due to the large interfacial surface and the low alginateconcentration. Further studies will be carried out in the future withincreasing concentrations of this polymer in the O/W HIPRE continuous

phase could be useful to determine the influence of alginate in theexternal phase of HIPREs.

In the same way that the studies performed with KP, the hydrophilicdrug CH was solubilized in W/O HIPREs (0.25 wt%) and O/W HIPREs(1.5%). CH is freely soluble in the receptor solution, then, sink condi-tions are accomplished in PBS because drug diffusion is not limited bylow solubility in the receptor solution. Fig. 9 shows the profiles of CHreleased to the PBS receptor solution as a function of HIPRE type andcomposition.

The results obtained show a low amount (around 20%) of CH re-leased from W/O HIPREs after 1440 min. These data are in agreementwith previous results obtained with W/O HIPREs and clindamycin hy-drochloride reported in the literature [9]. Initially, a limited burst isproduced (attributed to experimental factors such as droplet rupture byfriction when filling the dialysis bag), after which release of the drugreaches a plateau. CH is retained in the internal phase due to its highwater solubility. Drug solubility in the internal phase influences drugrelease more than the interfacial properties. In our experiments, theinfluence of alginate or crosslinked alginate is not significant comparedto the slow release influenced by the high solubility of the drug and therelease pattern is similar to those reported in the literature for thismolecule from W/O HIPREs [8]. In contrast to W/O HIPREs, when CHis solubilized in O/W HIPREs, the release is much faster but, as in thestudies performed with KP and described above, the influence of algi-nate or crosslinked alginate is negligible probably due to its low con-centration in the continuous phase of the emulsion and further ex-periments with a higher amount of alginate are needed to elucidate thepossible influence of alginate or crosslinked alginate in the continuousphase.

4. Conclusions

Biocompatible W/O and W/O HIPREs have been formed at 25 °C, insystems consisting of aqueous component/Cremophor W07/liquidparaffin and aqueous component/Cremophor RH455/Miglyol 812, re-spectively. In both kinds of emulsions the concentration of dispersephase was 83% and the aqueous component consisted of water, 1%sodium alginate solution or a mixture of sodium alginate solution andcalcium chloride solution (crosslinked calcium alginate). Droplet sizesof the W/O HIPREs formed were in the nanometric range, being smallerthan other HIPREs described in the literature. Ketoprofen, a lipophilicanti-inflammatory drug; or clindamycine hydrochloride, a hydrophilicantibiotic, were solubilized in both kinds of HIPREs and the influence ofthe composition of the aqueous phase on HIPREs stability and drugrelease was studied. Droplet sizes of the W/O HIPREs formed were inthe nanometric range, being smaller than other HIPREs described in theliterature. Rheology behavior studies showed an increase in the elasticmodulus (G’) of the emulsions in the presence of alginate or crosslinked

0

10

20

30

40

50

60

70

80

90

0 200 400 600 800 1000 1200 1400 1600 1800

HIPRE 1st alginate (HWOACKP)

HIPRE 1st calcium (HWOCAKP)

Fig. 7. Release profiles of KP from W/O HIPREs with different order of incorporation ofsodium alginate solution and calcium chloride solution.

0

20

40

60

80

100

0 100 200 300 400 500

0.36%KP in PBS1.5%KP in MiglyolFastum GelHIPRE HOWKPHIPRE HOWacKP

Fig. 8. Release profiles of KP from O/W HIPREs with different aqueos component com-positions, from a PBS solution, from a Miglyol solution and from a commercial gel.

0

20

40

60

80

0 200 400 600 800 1000 1200 1400 1600

HIPRE W/O water (HWOCH)HIPRE W/O alginate (HWOACH)HIPRE W/O alginate-calcium (HWOacCH)HIPRE O/W water (HOWCH)HIPRE O/W alginate (HOWACH)HIPRE O/W alginate-calcium (HOWacCH)

Fig. 9. Release profiles of CH from W/O and O/W HIPREs with different aqueous phasecompositions.

P. Bonilla et al. Colloids and Surfaces A 536 (2018) 148–155

154

alginate, and the influence of the order of incorporation of alginate andcalcium chloride in the viscosity was demonstrated, as an indication ofcrosslinking efficiency. KP release was slower when crosslinked alginatewas formed in the internal phase of W/O HIPREs. In contrast the in-fluence of crosslinked alginate on the release of CH was negligible andthe solubility of the drug in the disperse phase demonstrated to be themain mechanism responsible of CH release, as reported in the literaturein other studies with HIPREs. The results obtained suggest that theformation of a crosslinked alginate matrix in the internal phase ofHIPREs can have a striking influence on drug release and it has to betaken into account to develop controlled drug delivery systems. Thecharacteristics and differences in drug release profiles depending on thecomposition of the aqueous phase suggest the possibility of obtainingcontrolled release systems from crosslinked alginate highly con-centrated emulsions.

Acknowledgements

Financial support from Generalitat de Catalunya (grant2014SGR1655) and MINECO (grant CTQ2016-80645-R) is acknowl-edged. P. Bonilla acknowledges the Secretaría Nacional de EducaciónSuperior Ciencia y Tecnología SENESCYT Ecuador for their academicscholarship support.

References

[1] C. Solans, J. Esquena, N. Azemar, C. Rodríguez, H. Kunieda, Highly concentrated(gel) emulsions: formation and properties, in: D.N. Petsev (Ed.), Emulsions:Structure, Stability and Interactions, Elsevier, Amsterdam, 2004, pp. 511–555.

[2] H.M. Princen, A.D. Kiss, Osmotic pressure of foams and highly concentratedemulsions. 2. Determination from the variation in volume fraction with height in anequilibrated column, Langmuir 3 (1987) 36–41.

[3] C. Solans, R. Pons, H.T. Zhu, H.T. Davis, D.F. Evans, K. Nakamura, H. Kunieda,Studies on macro- and microstructures of highly concentrated water-in-oil emul-sions (gel emulsions), Langmuir 9 (1993) 1479–1482.

[4] G. Caldero, M.J. García-Celma, C. Solans, M. Plaza, R. Pons, Influence of compo-sition variables on the molecular diffusion from highly concentrated water-in-oilemulsions (gel-emulsions), Langmuir 13 (1997) 385–390.

[5] S. Rocca, M.J. García-Celma, G. Calderó, R. Pons, C. Solans, M.J. Stébé, Hydrophilicmodel drug delivery from concentrated reverse emulsions, Langmuir 14 (1998)6840–6845.

[6] G. Calderó, M.J. García-Celma, C. Solans, M.J. Stébé, J.C. Ravey, S. Rocca, R. Pons,Diffusion from hydrogenated and fluorinated gel-emulsion mixtures, Langmuir 14(1998) 1580–1585.

[7] G. Calderó, M.J. García-Celma, C. Solans, R. Pons, Effect of pH on mandelic aciddiffusion in water in oil highly concentrated emulsions (gel-emulsions), Langmuir16 (2000) 1668–1674.

[8] G. Calderó, M. Llinàs, M.J. García-Celma, C. Solans, Studies on controlled release ofhydrophilic drugs from W/O high internal phase ratio emulsions, J. Pharm. Sci. 99(2010) 701–711.

[9] G. Calderó, A. Patti, M. Llinàs, M.J. García-Celma, Diffusion in highly concentrated

emulsions, Curr. Opin. Colloid Interface Sci. 17 (2012) 255–260.[10] M. Llinàs, G. Calderó, M.J. García-Celma, A. Patti, C. Solans, New insights on the

mechanisms of drug release from highly concentrated emulsions, J. ColloidInterface Sci. 394 (2013) 337–345.

[11] C. Solans, A. Pinazo, G. Calderó, M.R. Infante, Highly concentrated emulsions asnovel reaction media, Colloids Surf. A: Physicochem. Eng. Aspects 176 (2001)101–108.

[12] L. Espelt, P. Clapés, J. Esquena, A. Manich, C. Solans, Enzymatic carbon-carbonbond formation in water-in-oil highly concentrated emulsions (Gel emulsions),Langmuir 19 (2003) 1337–1346.

[13] W. Busby, N.R. Cameron, C.A. Jahoda, Emulsion-derived foams (PolyHIPEs) con-taining poly(epsilon-caprolactone) as matrixes for tissue engineering,Biomacromolecules 2 (2001) 154–164.

[14] W. Busby, N.R. Cameron, C.A. Jahoda, Tissue engineering matrixes by emulsiontemplating, Polym. Int. 51 (2002) 871–881.

[15] C. Canal, R.M. Aparicio, A. Vílchez, J. Esquena, M.J. García-Celma, Drug deliveryproperties of macroporous polystyrene solid foams, J. Pharm. Pharm. Sci. 15 (1)(2012) 197–207.

[16] T.L. Hwang, C.L. Fang, C.H. Chen, J.Y. Fang, Permeation enhancer-containingwater-in-oil nanoemulsions as carriers for intravesical cisplatin delivery, Pharm.Res. 26 (10) (2009) 2314–2323.

[17] N. Fa, V.G. Babak, M.J. Stébé, The release of caffeine from hydrogenated andfluorinated gel emulsions and cubic phases, Colloids Surf. A 243 (2004) 117–125.

[18] V. Babak, M.J. Stébé, N. Fa, Physico-chemical model for molecular diffusion fromhighly concentrated emulsions, Mendeleev Commun. 13 (6) (2003) 254–256.

[19] R. Pons, G. Calderó, M.J. García-Celma, N. Azemar, I. Carrera, C. Solans, Transportproperties of W/O highly concentrated emulsions (gel-emulsions), Prog. ColloidPolym. Sci. 100 (1996) 132–136.

[20] T.S. Dunstan, P.D. Fletcher, Compartmentalization and separation of aqueous re-agents in the water droplets of water-in-oil high internal phase emulsions, Langmuir27 (2011) 3409–3415.

[21] J. Esquena, C. Solans, Highly concentrated emulsions as templates for solid foams,in: J. Sjöblom (Ed.), Emulsions and Emulsion Stability, Taylor and Francis, NewYork, 2006, pp. 245–262.

[22] J.M. Williams, D.A. Wrobleski, Spatial distribution of the phases in water-in-oilemulsions. Open and closed microcellular foams from cross-linked polystyrene,Langmuir 4 (1988) 4062–4067.

[23] J. Miras, S. Vílchez, C. Solans, J. Esquena, Chitosan macroporous foams obtained inhighly concentrated emulsions as templates, J. Colloid Interface Sci. 410 (2013)33–42.

[24] C.H. Goh, P.W.S. Heng, L.W. Chan, Alginates as a useful natural polymer for mi-croencapsulation and therapeutic applications, Carbohydr. Polym. 88 (2012) 1–12.

[25] G. Grant, E. Morris, D. Rees, P. Smith, D. Thom, Biological interactions betweenpolysaccharides and divalent cations: the egg-box model, FEBS Lett. 32 (1) (1973)195–198.

[26] J. Paques, E. van der Linden, C. Van Rijn, L. Sagis, Preparation methods of alginatenanoparticles, Adv. Colloid Interface Sci. 209 (2014) 163–171.

[27] C. Ouwerx, N. Velings, M.M. Mestdagh, M.A.V. Axelos, Physico-chemical propertiesand rheology of alginate gel beads formed with various divalent cations, Polym.Gels Netw. 6 (1998) 393–408.

[28] R. Pons, P. Erra, C. Solans, J.C. Ravey, M.J. Stébé, 1993. Viscoelastic properties ofgel-emulsions: their relationship with structure and equilibrium properties, J. Phys.Chem. 97 (1993) 12320–12321.

[29] C. Solans, R. Pons, H. Kunieda, Gel emulsions: relationship between phase beha-viour and formation, in: B.P. Binks (Ed.), Modern Aspects of Emulsion Science, TheRoyal Society of Chemistry, Cambridge, 1998, pp. 367–394.

[30] F. Roig, C. Solans, J. Esquena, M.J. García-Celma, Preparation, characterization,and release properties of hydrogels based on hyaluronan for pharmaceutical andbiomedical use, J. Appl. Polym. Sci. 130 (2013) 1377–1382.

P. Bonilla et al. Colloids and Surfaces A 536 (2018) 148–155

155