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Colloids and Surfaces B: Biointerfaces 111 (2013) 446– 452

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al hom epage: www.elsev ier .com/ locate /co lsur fb

usion behaviour of aquaporin Z incorporated proteoliposomesnvestigated by quartz crystal microbalance with dissipation (QCM-D)

uesong Lia,b, Rong Wanga,b,∗, Filicia Wicaksanaa,b, Yang Zhaoa,b, Chuyang Tanga,b,aume Torresa,c, Anthony Gordon Fanea,b

Singapore Membrane Technology Centre, Nanyang Technological University, Singapore 639798, SingaporeSchool of Civil and Environmental Engineering, Nanyang Technological University, Singapore 639798, SingaporeStructural and Computational Biology, School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore

a r t i c l e i n f o

rticle history:eceived 30 January 2013eceived in revised form 28 May 2013ccepted 4 June 2013vailable online 26 June 2013

eywords:quaporin Ziomimetic membraneroteoliposome fusionCM-D

a b s t r a c t

Aquaporin-based biomimetic membranes have potential as promising membranes for water purificationand desalination due to the exceptionally high water permeability and selectivity of aquaporins. However,the design and preparation of such membranes for practical applications are very challenging as therelevant fundamental research is rather limited to provide guidance. Here we investigated the basiccharacteristics and fusion behaviour of proteoliposomes incorporated with aquaporin Z (AqpZ) on tosolid surfaces. This study is expected to offer a better understanding of the properties of proteoliposomesand the potential of the vesicle fusion technique. Our results show that after incorporation of AqpZ,the size and surface charge density of the proteoliposomes change significantly compared with thoseof liposomes. Although the liposome could easily form a supported lipid bilayer on silica via vesiclerupture, it is much more difficult for proteoliposomes to fuse completely into a bilayer on the samesubstrate. In addition, the fusion of proteoliposomes is further hindered as the density of incorporatedAqpZ is increased, suggesting that proteoliposome with more proteins become more robust. However,

both the liposome and proteoliposome have difficulty forming supported lipid bilayers on the surface ofa polyelectrolyte layer even though it carries an opposite charge, indicating that the polymer may playan important role in stabilising vesicles. It was also observed that a high concentration of AqpZ could beincorporated into the 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) liposome even though itspermeability decreased. These findings may provide some useful guidance for preparing such biomimeticmembranes.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

Aquaporins are water channel proteins that possess the func-ion of facilitated transport of water molecules while selectivelyejecting other species in biological membranes [1,2]. Due tohese promising properties, the incorporation of aquaporins intoiomimetic membranes with a high loading density is expectedo provide higher water flux and salt rejection than conventional

everse osmosis (RO) membranes [3]. Thus, aquaporin-based mem-ranes have the potential for seawater desalination at closer to the

∗ Corresponding author at: School of Civil and Environmental Engineering,anyang Technological University, Singapore 639798, Singapore.el.: +65 6790 5327; fax: +65 6791 0676.

E-mail address: rwang@ntu.edu.sg (R. Wang).

927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2013.06.008

minimum energy, and this has attracted increasing interest world-wide [4–9].

One commonly suggested protocol to prepare a biomimeticmembrane is to form a protein-containing supported lipid/polymermembrane on a porous substrate [4–8], which acts as a selec-tive layer for separation. From several methods used to preparethese membranes, vesicle fusion is the most widely proposed tech-nique due to the ease of preparation and characterisation. Mostimportantly, this technique is more suitable for incorporation ofmembrane proteins. Like many other membrane protein, aquapor-ins need a biocompatible environment similar to a cell membraneto exhibit their function. To date only a few lipids and amphiphilicblock polymers have proven to be biocompatible with aquaporins

[1,2,5,8]. In polar solvents, these lipids or polymers can assem-ble into a bilayer or a bilayer-like structure, where aquaporins canbe incorporated. Extensive studies have focused on the formationof supported lipid membranes by vesicle fusion on various solids

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10–12] and different theories have been proposed to elucidate theusion mechanisms of vesicles. Supported biomimetic membranesith proteins incorporated could also be prepared through prote-

liposome fusion on a solid surface [13–15]. However, it has beenound that certain membrane proteins could significantly affect theusion behaviour of proteoliposomes. Granéli et al. [16] reportedhat the water-exposed hydrophilic domains of membrane pro-eins in proteoliposomes could hamper the bilayer formation onilica, while the membrane proteins without a hydrophilic domainad little effect on it. Their work demonstrated that the fusionehaviour of certain proteoliposomes could be rather differentrom that of liposomes, depending on the structure of the integrated

embrane protein. A similar phenomenon was also observed in ourrevious study on the preparation of aquaporin Z (AqpZ)-basediomimetic membrane by vesicle fusion [8]. A proteoliposomeith a relatively high content of AqpZ could not completely fuse

nto a bilayer on the polymeric membrane surface even whent was subjected to a pressure that could efficiently assist theusion of liposomes. However, in contrast to Granéli’s findings,qpZ does not have a large hydrophilic domain [17]. Thus the fac-

ors that affect the fusion behaviour of proteoliposomes have noteen clearly identified due to limited fundamental research in thiseld.

It is widely known that direct exposure of membrane proteinso a solid surface would lead to immobilisation and reductionf their activity [13,16]. One common way to circumvent thisroblem is to insert a hydrophilic polymer layer between theilayer and the solid surface, which acts as a cushion to avoidirect contact of the substrate and proteins [18–22]. Commonlysed polymers include polyethylene glycol [23–25], polysaccha-ide [21,26], and polyelectrolytes [18,20,27,28]. Polyelectrolytesre the most widely used hydrophilic polymers for the supportsecause their opposite charges can enhance vesicle depositionnd facilitate their fusion on the polymer. Nevertheless, it waseported that the fusion behaviour of vesicles on some certain poly-ers was significantly different from that on hydrophilic solids

28,29].The quartz crystal microbalance with dissipation (QCM-D) tech-

ique is a powerful analytical tool that has been widely used tonvestigate the fusion behaviour of liposomes [10–12,30,31]. Thisevice involves real-time monitoring of resonant frequency, f, andnergy dissipation, D, to determine the mass and viscoelastic-ty of the adsorbed vesicles or bilayer, respectively. The changesf these two parameters can indicate whether the vesicle haseen fused into a bilayer or remains intact. The bilayer can bereated as a rigid thin film; hence the mass and viscoelasticityill change slightly when the vesicle fuses into a bilayer. If the

esicle stays intact upon deposition, the trapped water insidehe vesicle will cause substantial changes in frequency and vis-oelasticity. In addition to the QCM-D technique, other techniquesuch as atomic force microscopy (AFM) [28,32], surface plas-on resonance (SPR) [33,34], fluorescence microscopy [29], have

lso been employed to investigate the fusion behaviour of vesi-les.

In this present study, QCM-D was employed to study and com-are the fusion behaviour of proteoliposomes incorporated withqpZ on silica and a polyelectrolyte layer surface. In order tolucidate the effect of AqpZ content incorporated on the fusionehaviour, liposomes and proteoliposomes with different AqpZ-o-lipid ratios were prepared and characterised under the sameonditions. In addition to QCM-D, permeability, size and zeta poten-ial measurements were also performed to further explore the

usion behaviour of the proteoliposomes. It is expected that thistudy can shed light on the fundamentals of proteoliposome’susion on different surfaces, ultimately facilitating the preparationf biomimetic membranes.

interfaces 111 (2013) 446– 452 447

2. Materials and methods

2.1. Materials

1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) waspurchased from Avanti Polar Lipids (Alabaster, AL). DPhPC does notpresent a detectable gel to liquid crystalline phase transition overa large temperature range (−120 ◦C to +120 ◦C) [35]. Monopotas-sium phosphate, potassium chloride, and disodium phosphatewere acquired from Merck Chemical (Singapore). Detergent 1-n-octyl-�-d-glucopyranoside (OG) and sodium dodecyl sulfate(SDS) were purchased from Calbiochem® (Singapore) and Bio-Rad(Singapore), respectively. All other chemicals or materials werepurchased from Sigma–Aldrich (Singapore) unless otherwisestated. All chemicals were used without further purification.Milli-Q water (Millipore, integrated ultrapure water system) witha resistivity of 18.2 M� cm was used. A phosphate buffer with apH value of 7.8 was prepared with 2.68 mM KCl, 8 mM Na2HPO4and 1.5 mM KH2PO4 in Milli-Q water.

2.2. Liposomes preparation

The DPhPC lipid in chloroform was dried by a N2 stream andthen vacuumed overnight. The dried lipid film was rehydrated inthe phosphate buffer. After stirring for several minutes and threefreeze-thawing cycles, the suspensions were extruded 21 timesthrough a polycarbonate membrane with a mean pore size of200 nm for further use. The lipid suspension was extruded at 22 ◦C.

2.3. Aquaporin reconstitution into liposomes

A certain amount of AqpZ solution, according to the theoreti-cal protein-to-lipid ratio (PLR), was added to the DPhPC liposomesolution with 1 wt% OG. After incubation for 1 h, 0.05 g biobeadswere added into 1 ml liposome/aquaporin solution and rotated for1 h. An additional 0.15 g biobeads were then added and rotated foranother 1.5 h to remove the OG. In order to minimise the adsorp-tion of lipids by biobeads, all biobeads were incubated in the sameliposome solution for 1 h prior to addition for detergent removal.The proteoliposome solution was then extruded through a poly-carbonate membrane with a mean pore size of 200 nm by 11 timesfor further use. Extrusion experiments were performed at 22 ◦C.

2.4. Characterisation of liposomes and proteoliposomes

The size and zeta potential of liposomes and proteoliposomeswere characterised by zetasizer Nano ZS (Malvern, UK) at 22 ◦C. Thepermeability of liposomes and proteoliposomes was determined bya stopped-flow apparatus (SX20, Applied Photophysics) at 22 ◦C.In this process the liposome and proteoliposome were rapidlymixed with hyperosmolar phosphate buffer containing 600 mMsucrose. The difference in intravesicular and extravesicular osmo-larity rapidly induced the shrinkage of the vesicles. The shrinkagerates of liposomes and proteoliposomes were measured by lightscattering. Each stopped flow experiment gave five light scatteringtraces, which were subsequently averaged. The initial rising rate (k)was calculated by fitting the raw data of the averaged trace withOrigin software. The water permeability of liposomes, Pf (�m/s),was calculated using the following equation:

Pf = k

S/V × Vw × �osm(1)

where S/V is the ratio of the initial surface area to the volume ofvesicles, Vw is the partial molar volume of water (18 cm3 mol−1) and�osm (osmol/L) is the osmolarity difference between the intraves-icular and extravesicular solutions.

448 X. Li et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 446– 452

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ig. 1. (A) Normalised stopped-flow light scattering measurements of liposome anifferent PLRs.

.5. Substrate preparation

Prior to each QCM-D measurement, the SiO2 sensors (Q-Sense)ere cleaned in a 2% SDS solution followed by a water rinse. The

ensor was then dried with nitrogen gas and cleaned in a UV/OzoneroCleaner (Bioforce) for 15 min.

.6. Quartz crystal microbalance with dissipation (QCM-D)

The QCM-D measurements were performed with a QCM-D sys-em from Q-sense (Göteborg, Sweden). The lipid concentration inll samples is 1.5 mmol/L. The resonant frequency (f) of the sensorepends on the total oscillating mass. The adsorbed mass of theigid layer can be determined by the Sauerbrey relation:

m = −C · �f

n(2)

here C is the mass sensitivity constant (17.7 ng cm−2 Hz−1 at = 5 MHz) and �f is the frequency change at the nth (n = 1, 3, 5. . .)armonic. The mass change can be correlated to the frequency shiftbtained from QCM-D measurements. The dissipation change (�D)s defined by

D = Elost

2�Estored(3)

here Elost is the energy lost during one oscillation cycle and Estoreds the total energy stored in the oscillator. The mass and viscoelas-icity of the adsorbed film can be estimated by using a combinationf frequency and dissipation information. However, the Sauerbreyquation cannot be applied to a soft film. Therefore, another modelVoight-Kelvin-based model) is required to quantify the mass andiscoelastic properties of the adsorbed film. All QCM-D measure-ents were performed at 22 ◦C. Each measurement was performed

t several independent overtones (n = 3, 5, 7, 9, 11) and recogniseds valid data only when all overtones presented the same trend. Fur-hermore, each measurement was performed at least three timeso confirm its reproducibility. The frequency and dissipation data inhe third overtone (n = 3, i.e., 15 MHz) were provided in all QCM-Desults.

. Results and discussion

.1. Characteristics of liposomes and proteoliposomes

Table 1 lists the size and zeta potential data of AqpZ incor-orated proteoliposomes. It shows that the surface charges ofroteoliposomes increased correspondingly with the increasef AqpZ concentration in the proteoliposomes, while the zeta

eoliposome (PLR 1/200). (B) The permeability values (Pf) of proteoliposomes with

potential of liposome was only −1.64 mv. This phenomenon isattributed to the negatively charged AqpZ at the buffer pH. The sizeof proteoliposome also increased depending on the concentrationof AqpZ incorporated. A similar phenomenon was also observedin another proteoliposome system [16]. Each group of proteolipo-some was also observed to be smaller than the liposome (∼170 nm,obtained from pure liposome treated with the same procedure inthe absence of AqpZ). While the exact mechanism is not clear, ithas been suggested that the size of vesicles prepared by extrusionthrough track-etched membranes is determined by the pressure,membrane pore size and lipid properties [36]. Unlike liposome,proteoliposome is a more complicated system that comprises lipidsand proteins. Apparently, the incorporated AqpZ had a major influ-ence on the physicochemical properties of the vesicles.

3.2. Activity of AqpZ

The activity of AqpZ in the proteoliposome was measured by thestopped-flow apparatus. Typical stopped-flow results of liposomeand proteoliposome can be seen in Fig. 1A. The shrinkage rate ofproteoliposome was significantly greater than the one of liposome,which could be attributed to the higher water permeation of AqpZ.The permeability is comparable to our previous reported value [8]indicating that the AqpZ maintained its intrinsic permeability inDPhPC lipids under the conditions tested in this study. However,the water permeability did not increase proportionally with theincrease in PLR. Upon reaching a certain PLR value, a decrease inwater permeability occurred with further increase in AqpZ concen-trations (Fig. 1B). Similar results have also been observed in otherAqpZ incorporated proteoliposomes [1,25]. It has been suggestedthat the method of incorporation might be responsible for the lowerreconstitution efficiency of aquaporins and lower permeability ata high PLR [2]. However, no report in the literature has confirmedthis yet.

3.3. Fusion behaviour of DPhPC liposomes on silica

Fig. 2 shows the adsorption of DPhPC liposome on silica inthe phosphate buffer. The initial buffer washing induced a minorchange of f and D, which may be due to the difference of water andbuffer in terms of viscosity and density. Upon addition of the lipo-some solution, the �f gradually decreased and reached saturationin approximately 3 min. The saturation value of resonant frequencyin the buffer solution was around −28.8 Hz. By taking into account

the �f caused by the buffer, the �f induced by liposome adsorp-tion was about −27.6 Hz, which is close to other reported valuesfor the bilayer [11,16]. The minor discrepancy can be attributedto variations in the hydration mechanisms of the lipid bilayer in

X. Li et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 446– 452 449

Table 1Sizes and zeta potentials of proteoliposomes with different PLRs.

Vesicle Mean size (diameter, nm) Polydispersity index (PDI) Zeta potential (mv)

Proteoliposomes (PLR 1:400) 118.9 0.101 −4.5Proteoliposomes (PLR 1:200) 111.4 0.111 −5.1Proteoliposomes (PLR 1:100) 122.2 0.137 −9.7Proteoliposomes (PLR 1:50) 156.7

Proteoliposomes (PLR 1:25) 161.6

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proteoliposome (PLR 1/25) could not be entirely attributed to the

ig. 2. Changes in resonant frequency and dissipation versus time for exposure ofilica to a DPhPC liposome solution.

arious solutions or incomplete formation of a bilayer [16]. Uponater rinsing the �f increased and �D decreased, but their changesere almost the same as induced by buffer rinsing at the begin-ing. If there were many unruptured liposomes on the surface, thesmotic shock caused by water rinsing would have helped ruptureiposomes, which would induce a big change in �D and �f. How-ver, this was not observed in the present study, suggesting mostiposomes ruptured spontaneously upon interaction with silica andormed bilayers completely on the silica surface [37]. However, theissipation value for the bilayer (∼1.6 × 10−6) after buffer rinse waselatively higher when compared to other reported values of lipidilayers [11,38]. It is speculated that the high viscoelasticity maye induced by the unique structural features of phytanoyl lipids or

ncomplete fusion of few vesicles [39].

.4. Fusion behaviour of proteoliposomes with different PLRs onilica

As stated above, proteoliposome is relatively different from lipo-ome because the incorporated membrane protein can significantlyffect the properties of vesicles. In this present study, the vesicleusion could be governed by the size, the surface charge and theomposition of vesicles. In order to further investigate the effectsf AqpZ on vesicle fusion, proteoliposomes are divided into tworoups based on their sizes and zeta potential values. Since eachroup obtained similar vesicle sizes and zeta potential values, asuch the proteoliposomes with lower PLRs (1/400, 1/200 and 1/100)re categorized as group A, while the rest of the proteoliposomesre classified as group B. Fig. 3 presents the QCM-D results of bothroups (A and B). Unlike the liposome, the resonant frequency ofroteoliposomes in group A initially decreased. Upon reaching a

inimum value, the frequency then increased to an equilibrium

evel. As for the dissipation values, an opposite trend was observed.he dissipation initially increased, and then decreased after reach-ng a maximum value before finally approaching an equilibrium

0.149 −14.40.128 −23.5

level. This fusion behaviour was consistent with one pathway ofvesicle fusion [30]: the deposition of vesicles onto a surface induceda high mass and viscoelasticity change. When they reached a crit-ical vesicular coverage upon adsorption, the vesicles ruptured andreleased the trapped water causing the mass and viscoelasticityto decrease. In contrast to liposome, which directly ruptures uponinteraction with silica, proteoliposome may need to overcome anenergy barrier to fuse. Although these proteoliposomes have a sim-ilar fusion pathway, some slight variations exist between them. Atcritical vesicular coverage, proteoliposomes with higher PLR obtaina lower f value and a higher D value. It is believed that the minordifferences of size [12] and surface charge [32] of proteoliposomein group A have little effect on the shifting of frequency and dissi-pation, therefore, it is more difficult for the proteoliposome with ahigher PLR to rupture on the silica surface. Furthermore, proteoli-posome with higher PLRs exhibited lower and higher equilibriumvalues of f and D, respectively. In addition, these two parame-ter values of all proteoliposome samples were much higher thanthose of a bilayer formed by liposome. Such phenomenon is alsoobserved in other proteoliposome systems [16]. For certain PLRs, itis estimated that AqpZ could supply around −9 Hz when proteoli-posome with PLR 1/100 forms a supported bilayer. The dissipationshift should not be caused by the AqpZ as proteins have littlecontribution to produce substantial dissipation change on a solidsurface [40,41]. The presence of unfused vesicles or semi-fusedvesicles on silica surfaces has also been reported. It is further statedthat proteoliposome carrying more proteins is considerably moredifficult to fuse into a bilayer. As the surface charge of proteolipo-somes did not vary significantly, the only possible reason could beassociated with the incorporated AqpZ molecules. It was proventhat the hydrophilic parts of several specific proteins such as pro-ton translocating nicotinamide nucleotide transhydrogenase (TH),could hamper the formation of bilayers for proteoliposomes [16].However, unlike proteins with large hydrophilic domains, aqua-porins are a group of highly hydrophobic membrane proteins withsmall hydrophilic parts and have a similar side length to the domainII of TH [42,43]. Therefore, the most probable reason for the inhi-bition of proteoliposome fusion would be the presence of AqpZwith vesicles of increased mechanical strength. AFM force indenta-tion tests have shown that the incorporated AqpZ could drasticallyimprove the mechanical stability of lipid membranes [25]. Theincreased mechanical strength of the lipid bilayer would increasethe energy barrier for bilayer formation through vesicle rupture.

The resonant frequency of group B gradually decreased until itreached an equilibrium level, while the D value was raised until itapproached an equilibrium level (Fig. 3). These equilibrium valuesare lower (for f) and higher (for D) than those of group A. As proteoli-posomes in group B carry more negative charge than those in groupA, the surface charge should be taken into account as more negativecharge could inhibit the fusion of vesicle on a negatively chargedsilica [32]. However, when compared to other reported values, sucha significant decrease of frequency and increase of dissipation for

surface charge [32,44]. It is believed that the improved mechanicalstability also contributed to the presence of unfused proteolipo-somes. Although the proteoliposome with PLR 1/25 had a lower

450 X. Li et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 446– 452

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some in the phosphate buffer may be responsible for the presenceof unfused and loosely bound vesicles as the electrostatic forcebetween the vesicles and surface is the main driving force to forma bilayer [37].

Fig. 3. (A–B) The changes in frequency and dissipation of QCM-D for

ermeability than that with PLR 1/50, it seems the proteoliposomeith PLR 1/25 carried more aquaporins based on the QCM-D results.

hus, it should be determined whether the lower permeability ofhe proteoliposome with the higher PLR was caused by a lowerncorporation efficiency of AqpZ. As mentioned previously, the pro-eoliposome with PLR 1/25 exhibited a higher mechanical stabilityhan other proteoliposomes due to a higher loading density of pro-eins incorporated. Furthermore, a higher surface charge densityorresponds to a higher density of AqpZ molecules. Therefore, theecrease in the intrinsic water permeability of AqpZ should note due to the lower incorporation efficiency. Liposome could still

ncorporate a higher density of AqpZ beyond the optimal PLR ratio.ne possible reason for the decrease in water permeability of theroteoliposome is that strong interactions among AqpZ moleculest a high protein density have inhibited their function.

In order to compare the degree of fusion of individual pro-eoliposome groups, a bilayer coverage is estimated from thequilibrium dissipation value based on the method described byranéli [16]. The corrected equations are as follows [16]:

= 1 − �Dm − �Db

�Ds − �Db(4)

nd

Ds = ̌ · d (5)

here �Ds is the dissipation change at saturation when the surfaces fully covered by intact vesicles, �Dm is the actual measured dissi-ation change at saturation, �Db is the dissipation change of bilayerbtained for the liposome, which is about 0.1 × 10−6 [16,32]. d is theiameter of vesicle and ̌ is the constant ratio of �Ds to the vesicleiameter, equal to 0.15 [12,16]. The bilayer coverage versus PLR isiven in Fig. 4. Interestingly, the bilayer coverage decreases withhe increase in concentration of AqpZ incorporated, indicating thathe incorporated AqpZ has inhibited the fusion of proteoliposomes.

.5. Fusion behaviour of liposome and proteoliposome onolymer surface

Polydiallyl dimethyl ammonium chloride (PDADMAC) wasissolved in the phosphate buffer and deposited on the sil-

ca surface. QCM-D results show that the deposited PDADMAC

me and the proteoliposomes with different PLRs adsorption on silica.

layer only caused minor changes in frequency (∼−9.75 Hz) anddissipation (∼1.38 × 10−6), suggesting that this layer was very thinand rigid. Fig. 5 depicts QCM-D results for liposome and prote-oliposomes depositions on a PDADMAC layer. In contrast to thefusion behaviour of liposome on silica, considerable decrease infrequency and increase in dissipation occurred throughout a verylong deposition process. This indicates that the liposome depositedgradually and remained intact on the PDADMAC layer. Similarphenomena were obtained from the QCM-D measurements ofzwitterionic liposomes deposition on other polyelectrolyte layers[27,28]. Significant increase in frequency and considerable decreasein dissipation occurred upon buffer rinse before reaching a stablelevel, indicating that a large portion of the loosely bound vesicleshad been washed away by the buffer. A low charge density lipo-

Fig. 4. Bilayer coverage formed by proteoliposomes on silica surface as a functionof PLR. (These error bars were obtained from three independent overtone measure-ments).

X. Li et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 446– 452 451

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ig. 5. The changes in frequency and dissipation of QCM-D for (A) liposome, (B) proteayer.

In contrast to a long equilibrium time (more than 1 h) needed foriposome deposition, the proteoliposomes only required a few min-tes to reach a stable state after depositing on the PDADMAC layerFig. 5B and C), signalling a strong affinity between the proteoli-osome and the polyelectrolyte, probably attributed to the greateregative charge density of the proteoliposome. However, a suddenecrease of resonant frequency and increase of dissipation changesf proteoliposome with PLR 1/200 were observed upon depositionf proteoliposomes. Once reaching a minimum/maximum level, amall increase in f and decrease in D occurred within a short periodf time before finally reaching an equilibrium level (Fig. 5B). Asescribed above, this phenomenon has been commonly found inesicle fusion through a critical vesicular coverage. However, thequilibrium f and D values have shifted greatly as compared tohe values of a bilayer, indicating that the majority of proteolipo-omes remained intact after interacting with the polyelectrolyteayer though a small portion of vesicles have fused. However, vesi-le fusion was not observed for the proteoliposomes with PLR/25, since the f and D gradually approached equilibrium levels.or liposome, the increase in surface charge could have facilitatedts rupture and fusion on a polyelectrolyte layer [28]. Nonethe-ess, it was not the same case for proteoliposome in the presenttudy. Although the proteoliposomes with PLR 1/25 carry a higherharge density, which should help vesicles fuse on charged poly-er surface, fewer vesicles were fused on the polymer layer as

ompared to the proteoliposomes with PLR 1/200. As discussedbove, an improved mechanical stability of vesicles by AqpZ mightnhibit the vesicle fusion. Despite the electrostatic force betweenhe proteoliposome and the polyelectrolyte layer that could facil-tate vesicle fusion, the effect of increased mechanical strength ofesicle appears to be much more significant. In our previous study,t was observed that proteoliposomes with a higher protein densityecame more difficult to fuse on a polymer surface even when itas conducted under applied pressure, though the pressure could

reatly facilitate the fusion of liposomes [8]. It is noticeable thathe equilibrium frequency value for the proteoliposomes with PLR

/200 or PLR1/25 on the PDADMAC layer was considerably higherhan that of the same proteoliposomes on silica. Since the equi-ibrium frequency change was dominated by the mass trappedn the sensor surface, the water inside and between the vesicles,

ome with PLR 1/200 and (C) proteoliposome with PLR 1/25 deposition on PDADMAC

only slight deformation of proteoliposome on the polymer sur-face occurred, resulting in an increase of effective thickness [12].This suggests that these deposited vesicles became less deformedat the equilibrium state. One probable explanation for this is thatthe polyelectrolyte played an important role in stabilising the vesi-cles, which was also found in other vesicle-polyelectrolyte systems[27,28]. It appears that the stabilisation effect by polyelectrolyte isnot negligible.

4. Conclusions

Both zeta potential and QCM-D results reveal that a highconcentration of AqpZ could be incorporated into the DPhPC lipo-somes ever though its permeability would decrease as indicatedby stopped-flow tests. However, it is much more difficult for theAqpZ incorporated proteoliposome to fuse into a bilayer than lipo-somes on silica surfaces, especially with an increase in the densityof AqpZ in the proteoliposome. The increased surface charge andmechanical strength of the proteoliposome may affect the fusionand hinder the bilayer formation. The QCM-D results also indi-cate that the proteoliposome can remain intact and is stable ona polyelectrolyte surface. Since the vesicle fusion method has beenwidely employed to prepare supported lipid membranes, furtheroptimisation is needed to facilitate proteoliposome fusion in orderto improve the quality of the bilayer. With the increasing interestin aquaporin-based biomimetic membranes for water purificationapplications, the findings from our work can provide importantguidance for designing such biomimetic membranes as well as forbetter understanding of the biomimetic system.

Acknowledgments

This research grant is supported by the Singapore NationalResearch Foundation under its Environmental & Water Tech-nologies Strategic Research Programme and administered by the

Environment & Water Industry Programme Office (EWI) of the PUB(MEWR 651/06/169). We are also grateful to Singapore EconomicDevelopment Board for funding to Singapore Membrane Technol-ogy Centre.

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