Biomimetic membrane for desalination and water reuse 2

30 Chapter AquaporinZ Incorporation via Binary-Lipid Langmuir Monolayers 2.1 Introduction LB technology is known to be a powerful method to study lipid-protein interaction at the gas-liquid interface and form planar lipid bilayers Membrane insertion behavior of water soluble proteins is commonly investigated on the LB surface to mimic biological membranes However, hydrophobic membrane proteins like AqpZ which have to be solubilized in a detergent solution are poorly studied with the LB technique This is mainly due to the fact that the detergent can easily disrupt the surface monolayer by either adsorbing onto the monolayer or solubilizing the monolayer [92, 93] As a result, the process of membrane protein insertion is hindered by the detergent-monolayer interaction Moreover, the incorporated protein has the tendency to denature and lose its activity when it reaches the air-liquid interface [60] All of these reasons make the incorporation of membrane proteins with the LB method a challenging and intriguing work In this study, we attempt for the first time to reconstitute AqpZ into the lipid bilayer with the LB technique As detergent removal is critical for the membrane protein incorporation as well as maintaining the membrane stability and integrity, the first goal of this work aims to prove that the detergent adsorption on the lipid monolayer can be suppressed through the addition of BioBeads in the subphase, and the detergent removal rate is correlated to the amount of BioBeads and circulation in the subphase For the second part of this work, we demonstrated a new AqpZ incorporation approach with a binary-lipid Langmuir monolayer and propose a three- 31 step mechanism for protein incorporation As atomic force microscopy (AFM) is the unique method to characterize LB film in the nano-scale [94, 95], it was adopted for the membrane morphology study in this work 2.2 Materials and methods 2.2.1 Materials Nickel-chelating lipids, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-(N-(5-amino-1- carboxypentyl)iminodiacetic acid)succinyl with Nickle (DOGSNTA) and 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Avanti Polar Lipids, Birmingham, United States n-dodecyl beta-D-maltoside (DDM) with purity > 99.5% was a product of Acros Organics, Geel, Belgium 10x Phosphate buffered saline (PBS) in ultra high purity (1st BASE, Singapore) was diluted 10 times with Milli-Q water (Millipore) before use (pH=7.4) The lipids solution was prepared in chloroform (Tedia, HPLC grade) at a concentration of 1mg/ml Bio-Beads SM-2 Absorbents from BIO-RAD was used to remove DDM in the subphase Grade V1 mica was purchased from SPI Supplies 2.2.2 Surface pressure measurement The surface pressure measurement was carried out on a customized LB trough (KSVNIMA, Finland) with a trough area of 36x5 cm2 Before each experiment, the trough was wiped thoroughly with chloroform twice and subsequently rinsed with Milli-Q water The subphase in the trough was either Milli-Q water or PBS buffer solution, and all the experiments were performed at controlled room temperature (23±1°C) Surface pressure was measured by a pressure sensor based on the Wilhelmy plate method The effective surface area was controlled by a pair of Delrin barriers Lipid 32 solutions were spread on the aqueous subphase when the barriers were opened to the maximum and 60min was allowed for chloroform evaporation from the surface The total volume of the subphase is 65±2 ml For injection of DDM or AqpZ solution into the subphase, barriers were held statically after the surface pressure reached 30mN m-1 An injection port located at the center of the trough was utilized to reduce the disturbance on surface monolayer during injection After injection, the subphase was circulated at 3ml/min to promote equilibration for hours and the change in surface pressure was recorded In the case of detergent removal from the subphase, BioBeads were added into the dipping well before spreading of the monolayer and stirred gently with a mini Teflon stir bar Subphase circulation was performed throughout the entire detergent removal process The air above the subphase was maintained at saturation level to limit evaporation of water To obtain the pressure-area isotherms of lipids monolayer, the surface was compressed or expanded at the rate of 10cm2/min All the experiments were repeated for at least three times to ensure the reproducibility of the results 2.2.3 Surface tension measurement A surface pressure sensor was used to determine the surface tension of detergent/protein solutions A platinum Whilhelmy plate connected to the pressure sensor was partially immersed in a 20ml PBS solution The surface pressure change (π) of PBS buffer with changing DDM concentration in the buffer was measured by the pressure sensor 33 The surface tensions of mixtures were calculated and plotted against DDM concentration Three times repetition were done for each data point and the error for all points are within 6% 2.2.4 LB film deposition and AFM scanning LB film deposition was performed to transfer the monolayer from the air-liquid interface to the mica surface The surface pressure for deposition was always at 35mN m-1 To study the morphology of the DPPC layer with DDM disruption and BioBeads intervention, the DPPC monolayer was deposited onto a fresh cleaved mica surface first by the vertical LB method and followed with the horizontal LS method The transfer speed for the LB method is mm min-1 The samples were immersed in Milli-Q water before and during AFM scanning In the case of protein-associated monolayer deposition, a first monolayer of pure DPPC-DOGSNTA (5:1) was prepared by LB deposition on the mica surface using another trough (KSV-NIMA, Inverted Microscopy Trough) The protein-associated DPPC-DOGSNTA monolayer was subsequently deposited onto the first layer by the LS method The samples were incubated in PBS buffer for hour, and then imaged by AFM in PBS buffer environment The films were imaged by a PicoSPM atomic force microscope (Agilent) in the Acoustic alternating current mode The samples were scanned with ULTRASHARP NSC15/AIBS cantilevers (resonance frequency between 265 to 400 kHz and typical force constant of 46N m-1) in aqueous solutions (Milli-Q water or PBS buffer) at room temperature (23 ±1°C) The scanning speed is less than 0.5 ln s-1 34 2.3 Results and discussion 2.3.1 DDM effects on DPPC monolayer A well characterized lipid, DPPC, was selected in this experiment to demonstrate that detergent penetration into the lipid monolayer could be eliminated with the addition of BioBeads BioBeads are macroporous polystyrene beads that could remove detergent efficiently by hydrophobic adsorption At room temperature (23°C±1), DPPC at the air-water interface exhibits a liquid-condensed phase upon compression to above mN m-1; therefore, detergent has the poor accessibility to such a compact monolayer [93] To avoid a heavy loss of surface lipids by detergent solubilization, the DDM concentration in the subphase was controlled at µM (2 mg L-1), which was much lower than its critical micelle concentration (CMC), 0.18 mM [96] As shown in the dashed lines of Figure 2.1, the isotherm cycle of the monolayer after the addition of DDM displays a significant counterclockwise hysteresis, while it is known that the pure DPPC monolayer does not show any hysteresis[97] The hysteresis is caused by the DDM adsorption onto the monolayer at low surface pressures and incomplete desorption from the monolayer at high surface pressures Different amounts of BioBeads (50mg, 150mg and 300mg, in wet weight) were introduced into the trough to study the influence of BioBeads addition on the detergent removal rate Since BioBeads is only confined in the dipping well (1.5cm in diameter) located at the center of the trough, effective detergent removal cannot be achieved without subphase circulation 35 Figure 2.1 Pressure-area isotherm cycles of DPPC at 23°C after hours detergent removal with (a) 50mg, (b) 150mg, and (c) 300 mg BioBeads in the trough (dotted line) and subphase volume of 65±2ml The initial DDM concentration in the subphase is mg L-1 The isotherm cycles after detergent removal are compared with pure DPPC isotherm (solid line) and isotherm cycle without using BioBeads (dashed line) The isotherm cycle of DPPC monolayer was obtained after hours of detergent removal The relative amount of remaining DDM in the subphase can be reflected by the intensity of isotherm-cycle hysteresis as well as the lowest surface pressure in the cycle When 50mg BioBeads was used (Figure 2.1(a)), the isotherm cycle shows similar intensity of hysteresis with the one that has no BioBeads, but the lowest surface pressure has been reduced to 0mN m-1 from 5mN m-1, indicating that only a small fraction of the detergent in the subphase has been removed A significant 36 improvement can be observed from the isotherm cycle as the BioBeads amount increased to 150mg (Figure 2.1(b)), with milder hysteresis and better proximity to the pure DPPC isotherm With 300mg BioBeads (Figure 2.1(c)), though the monolayer displays a gentle hysteresis, the shape of the compression isotherm has reverted to that of a pure DPPC isotherm Therefore, DDM can be eliminated from the interface at a faster rate when there is a higher BioBeads to DDM ratio, which is reflected by a better recovery of DPPC isotherms The effect of DDM on the DPPC monolayer was also demonstrated by AFM images in Figure 2.2 Without the addition of BioBeads, the DPPC bilayer loses the original compact structure (Figure 2.2(a)) and forms a “fluid-like” defective structure (Figure 2.2(b)), while with detergent removal by BioBeads, the compact bilayer structure (Figure 2.2(c)) was restored Therefore, it can be concluded that the detergent content at the air-liquid interface can be substantially reduced with a decrease in its subphase concentration BioBeads reduce the adsorbed DDM in the DPPC monolayer by removing the DDM in the bulk either before or after the DDM association with the monolayer In the former case, DDM adsorbs to BioBeads faster than to the monolayer; thus less detergent is available to disrupt the surface monolayer In the latter case, DDM quickly reaches the surface monolayer, but the elimination of DDM in the subphase leads to DDM desorption from the monolayer Both hypotheses may be reasonable but require further verification 37 Figure 2.2 AFM topograph of (a) pure DPPC bilayer, (b) disrupted DPPC bilayer by 2mg L-1 DDM in the subphase, and (c) DPPC bilayer with removal of subphase DDM All the images were scanned in ultrapure water environment The dark area corresponds to the mica surface The typical DPPC thickness should be around 4-5 nm as shown in (a) and (c) However with DDM disruption, the DPPC thickness is reduced below 4nm and the “fluid-like” defective structure is formed in the bilayer The cross-section profiles at the dashed lines are shown at the bottom of respective images The scale bars (in solid line) are 1µm in all the three images 45 Surface Pressure, mN/m 40 Pure DPPC 35 Pure DOGSNTA 30 5:1 (DPPC:DOGSNTA) 25 10:1 (DPPC:DOGSNTA) 20 15 10 40 50 60 70 80 90 100 110 120 Molecular Area, Å2 Figure 2.3 Pressure-area isotherms of DPPC, DOGSNTA and their mixtures of different molar ratios (5:1 and 10:1) The temperature of experiments is 23±1°C 38 2.3.2 AqpZ incorporation via DPPC-DOGSNTA monolayers Monolayers of DPPC and DOGSNTA mixture were produced by spreading the mixed lipid solution with certain ratio on the PBS buffer surface Figure 2.3 shows the pressure-area isotherms of pure DPPC, pure DOGSNTA and their mixtures of different molar ratios (5:1 and 10:1) The pure DOGSNTA monolayer has no liquidexpanded to liquid-condensed transition plateau in the isotherm showing that DOGSNTA exist in the fluid-phase at the room temperature In contrast, DPPC has a transition temperature of 40°C thus is in the gel-phase at the room temperature Therefore, their mixtures display characteristics of both To preserve the compact structure of the monolayer at high surface pressures, DPPC and DOGSNTA mixed at a ratio of to was adopted for protein insertion As mentioned previously, DPPC is able to form a closely packed monolayer at high surface pressures and resist the invasion of detergents In the meantime, DOGSNTA provides binding sites for Histagged proteins [98], which ensures the monolayer to have a good affinity to AqpZ Upon addition of DOGSNTA in the DPPC monolayer, the surface pressure at liquid expanded-liquid condensed (LE-LC) phase transition increases and the transition plateau becomes less prominent compared with the pure DPPC monolayer These suggest that the monolayer formed with the DPPC-DOGSNTA mixture possesses more fluidity than that formed with the pure DPPC The AqpZ-DDM solution was injected at a surface pressure of 30mN m-1 This surface pressure is selected for two reasons The first is to minimize the detergent penetration into the monolayer As described above, DDM can be partially desorbed (i.e squeezed out) from the tightly packed LC phase monolayer, which causes the 39 hysteretic feature in the isotherm cycle of DPPC/DDM Thus, at a high surface pressure of 30mN/m, less disruption of the monolayer by detergent is expected [99] Secondly, the transmembrane protein AqpZ has a great tendency to aggregate and be denatured when exposed to air Therefore, the densely packed monolayer would limit the exposure of proteins to air at the air-liquid interface As the protein-detergent mixture is injected into the DPPC-DOGSNTA subphase, one critical issue is that the lipid monolayer may be dissolved when the detergent concentration is above CMC [93, 100] This is especially true for DOGSNTA because it is in the fluid phase at room temperature and therefore has no liquid condensed phase on the air-liquid interface However, it is known that AqpZ needs to be stored at a detergent concentration higher than CMC to prevent aggregation The lowest association concentration, the critical aggregation concentration (CAC), of DDM in the presence of AqpZ in the system, was therefore studied by the surface tension method [101] With the presence of proteins or polyelectrolyte in the solution, complexation of the detergent with protein/polymer molecules is thermodynamically favored at the CAC rather than the formation of regular micelles [102, 103] The hydrophobic segment of proteins associates with hydrocarbon chains of detergents and forms a protein-surfactant complex As shown in Figure 2.4, at an AqpZ concentration of 0.5 mg L-1, the CAC of the mixed system is at mg L-1 DDM, which is well below the CMC value of pure DDM solution 85 mg L-1 [96] The existence of CAC proves that nonspecific hydrophobic interactions between DDM and AqpZ are strong enough to induce complex association of the two at a DDM concentration lower than CMC The protein-detergent complex formed at CAC has been named as a “necklace” model with detergent micelles as beads on the protein chain [103-105] 47 Figure 3.1 Schematic presentation of immobilization of the cross-linked proteoliposome on a PDA coated membrane (not to scale) 3.2 Materials and methods 3.2.1 Materials The lipids, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and the ammonium salt of 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(amino(polyethylene glycol)-2000) (DSPE-PEG-NH2) were purchased from Avanti Polar Lipids Ammonium salt of 1,2-dimyristoyl-sn-glycero-phosphoethanolamine-N-(lissamineRhodamine B sulfonyl), Rh-PE, was obtained from Invitrogen Ethylene glycol dimethacrylate (EGDMA), 1-Hydroxycyclohexyl phenyl ketone (Irgacure184), 6Carboxyflurescein (CF), dopamine hydrochloride, isopropyl alcohol (IPA), glutaraldehyde solution (50 wt% in H2O), chloroform, sodium chloride (NaCl) and magnesium chloride (MgCl2) were from Sigma-Aldrich N-methylpyrrolidone (NMP) was purchased from Merck The detergent, n-dodecyl beta-D-maltoside (DDM) was a product of Acros Organics 10x Phosphate buffered saline (PBS, pH=7.4) and sucrose in ultra high purity were purchased from 1st Base, Singapore Ultrapure water was produced by the Millipore Reference A+ system Bio-Beads SM-2 Absorbents and tris(hydroxymethyl) aminomethane (Tris) were purchased from BIO-RAD 48 Polyacrylonitrile (PAN) for substrate preparation was from Tong-Hua synthesis fiber Co Ltd (Taiwan) A 10-histidine residual tagged (His-tagged) The AqpZ stock solutions contain ~1mg/mL protein and 18mM DDM in PBS buffer 3.2.2 Preparation of Vesicles DOPC, DSPE-PEG-NH2, EGDMA and UV initiator Irgacure 184 were mixed at a molar ratio of 20:1:40:20 A multilamellar vesicle suspension in PBS buffer was prepared using the film rehydration method Small unilamellar vesicles (SUV) with a uniform pore size were produced by extruding the suspension through a polycarbonate Nuclepore track-etch membrane (Whatman) of 100 or 200 nm pore size For AqpZ reconstitution experiments, AqpZ stock solution was added during the film rehydration step and the mixture was agitated for at least hours Then, BioBeads were added into the mixture to remove the DDM completely The suspension was protected with purified argon and kept away from light source throughout the experiment Prior to the photo-polymerization of vesicles, the suspension was bubbled with purified argon to remove oxygen Radical polymerization was then initiated by incubating the suspension in a UV cross-linker (Vilber Lourmat, BLXE254, 245 nm, 40 W) for 30 The vesicle solution was maintained at a distance of 4cm from the UV bulbs 3.2.3 Characterization of polymerized vesicles To verify that the vesicles underwent polymerization, the DOPC/DSPE-PEGNH2/EGDMA/Irgacure184 mixture was doped with 0.1 wt% Rh-PE (excitation/emission= 560 nm/583 nm) before dry film formation In the film rehydration buffer, 0.01 mg mL-1 CF (excitation/emission= 494 nm/521 nm) was into 49 vesicles, and the suspension was used without extrusion The resultant suspension was separated into two batches, i.e with and without UV cross-linking Both batches of vesicles were dialyzed 48 hours against pure PBS buffer so as to remove the nonencapsulated CF The Rh-PE doped vesicles with CF encapsulation were imaged using a confocal laser scanning microscope (Nikon A1 Confocal) After depositing the suspension on the glass coverslip, confocal imaging was performed at two wavelengths simultaneously, 488 nm and 514.5 nm (both from an argon ion laser), to excite CF and Rh-PE, respectively It should take note that all the vesicles for confocal imaging were non-extruded 3.2.4 Vesicle permeability measurements The permeability of vesicles was determined using a stopped-flow apparatus (Chirascan, Applied Photophysics) By rapidly mixing the vesicle solution with a hypertonic buffer (0.6 mol L-1 sucrose), water would permeate outwards of the vesicles, causing them to experience a sudden shrinkage The rapid reduction of vesicle volume was recorded as an increase in the light scattering intensity at an emission wavelength of 577 nm To improve the signal to noise ratio, all the experiments were performed at a temperature of 8°C 3.2.5 Preparation of vesicle immobilized membranes Flat sheet PAN substrates were prepared by casting a 12 wt% PAN solution in NMP directly on glass plates with a 100 µm casting knife The membranes were then stored in ultrapure water until use PDA deposition on the top surface of PAN substrates was performed by mounting the membranes on a dead-end modification cell The dopamine solution (0.02 mg dopamine dissolved in 100 mL 10 mM Tris buffer with 50 pH=8.5) was added into the dead-end modification cell and stirred at 350 rmp for hours PDA deposition After deposition, the membranes were immersed in IPA for 30 to remove any unbound PDA and again rinse thoroughly with water The surface area of PDA-PAN membrane available for vesicle immobilization was 0.785 cm2 A 150 µL of the vesicle solution (6 mg mL-1) was filtered onto the membrane at a transmembrane pressure of 40 mbar for 10 The membranes were then incubated with the vesicle solution at 4°C for hours Afterwards, the vesicle solution was removed from membrane surface and 0.5 wt% aqueous glutaraldehyde was filtrated to cross-link the immobilized vesicles at a pressure of 100 mbar for 10 3.2.6 Field-emission scanning electron microscopy (FESEM) The morphology of freeze-dried membranes was observed by FESEM (JSM-6700F, JEOL) To image the membrane cross-section, the freeze dried samples were immersed in liquid nitrogen and fractured Before imaging, a layer of platinum was coated on the membrane using a JEOL JFC-1300 Platinum coater 3.2.7 Nanofiltration studies The nanofiltration tests were performed using dead-end permeation cells Pure water permeability, PWP (L m-2bar-1h-1) measurements were conducted at 23°C, bar, which was calculated using the equation below !"! = ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(5) !"# Q is the volumetric flow rate of water (L h-1), ∆P is the transmembrane pressure difference (bar) and A is the membrane surface area (m2) Rejections of NaCl and 51 MgCl2 solutions (200ppm in ultrapure water) were tested with a surface mixing speed of 700 rpm at 23°C, bar The membranes were flushed thoroughly with ultrapure water between tests of different solutes The salt rejection was calculated using the equation shown below ! % = !! − !! ×100!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(6) !! where Cf and Cp are the concentrations of the feed solution and the permeate, respectively Data were obtained from at least three different preparations 3.3 Results and discussion 3.3.1 Characterization of polymerized vesicles To study the polymerization of cross-linker EGDMA within the lipid bilayer, CF was encapsulated in the designed vesicles labeled with 0.1% Rh-PE Due to the high fluidity of DOPC bilayers, the CF could diffuse out of the bilayer if there was a concentration gradient Therefore, by reducing the CF concentration in the bulk solution via dialysis, the dye can be slowly removed from the hydrophilic core of the liposomes Using confocal laser scanning microscopy, CF and Rh-PE were excited at the same time to obtain an overlapped red and green fluorescence image, where the red refers to Rh-PE while the green refers to CF Figure 3.2(a) corresponds to the images of liposomes without UV polymerization, which did not display green fluorescence from both inside and outside the liposomes As expected, most CF experienced diffusion out of the liposome through the DOPC bilayers When a crosslinked polymer network was formed in the lipid bilayer, the green dye was trapped inside the red vesicles as shown in Figure 3.2(b) The formation of the hydrophobic network in the bilayer caused the vesicles to be much less permeable (or may be 52 impermeable) to hydrophilic molecules like CF, thus these hydrophilic molecules ended up being encapsulated within liposomes Figure 3.2 Confocal microscopy images of Rh-PE doped liposomes (a) without and (b) with UV cross-linking of the bilayer CF can diffuse through the control vesicles and be removed in dialysis process in (a), but with formation of a polymer network within the lipid layer, fluorescein was trapped inside the liposomes as shown in (b) Note: The vesicles here were not extruded.!! A field emission transmission electron microscopy (FETEM) was used to image DOPC/DSPE-PEG-NH2 vesicles with CF encapsulation Small unilamellar vesicles extruded from a membrane with an average pore size of 200nm were prepared Both cross-linked and non-cross-linked liposomes (1 mg mL-1) with CF encapsulation were subjected a day dialysis against PBS buffer The FETEM images are shown in Figure 3.3 Similar with the confocal imaging results, after cross-linking, CF were trapped inside the vesicle as shown in Figure 3.3(b) The bilayer thickness of the DOPC/DSPE-PEG-NH2 vesicles was estimated at around 15 nm from the TEM images The thickness of lipid bilayer is typically about nm However, in our case, the thickness is much higher probably because the PEG brushes were formed on both the lumen and shell sides of the vesicles, and the hydrophobic monomers might swell the bilayer to a certain extent 53 15nm% 100 nm (a) 100 nm (b) Figure 3.3 FETEM images of DOPC/DSPE-PEG-NH2 vesicles after extrusion from 100 nm (a) without and (b) with UV cross-linking of the bilayer The thickness of the vesicle wall is about 15nm from the images 3.3.2 Vesicle immobilization on PDA coated silicon surface To demonstrate the immobilization ability of our designed liposomes on PDA surfaces, AFM was adopted to study the conjugated liposomes on a PDA coated silicon substrate Prior to vesicle deposition, the silicon substrate was immersed in a 0.2 mg mL-1 dopamine solution for hours to obtain a thin layer of PDA on the surface and then rinsed with IPA to remove the loosely bounded PDA AFM scanning showed that the PDA coated silicon surface has a surface roughness of 0.71±0.12 nm Polymerized liposomes with a diameter of approximately 200 nm were incubated on the PDA coated silicon substrate for hours at 4°C Liposomes with mole% DSPEPEG-NH2 can actively bond with PDA through amine-catechol adduct formation In Figure 3.4, it was observed with the aid of AFM that circular spheres were scattered on the PDA coated silicon surface A representative cross-section of a spherical feature is shown in Figure 3.4 (c) To preserve the structure of conjugated liposomes, the sample was imaged in a wet environment (ultrapure water) The diameter of the spheres is ranged from 100 to 300 nm thus is corresponding to the size of the polymerized vesicles Height of these vesicles is lower than 200 nm probably because of a large contact area of the liposome with the PDA-silicon surface The films were 54 imaged by a PicoSPM atomic force microscope (Agilent) in the Acoustic alternating current mode The samples were scanned with OLYMPUS cantilevers (OMCLTR400PSA, resonance frequency of 11 kHz and typical force constant of 0.02 N m-1) in aqueous solutions (ultrapure water) at room temperature (23 ± 1°C) The scanning speed was less than 0.5 ln s-1 (a) (b) (c) Figure 3.4 AFM images of immobilized liposomes on PDA deposited silicon surface in amplitude graph (a), 3D topography (b), and a cross-sectional profile (c) of a representative liposome pointed by an arrow in (b) 3.3.3 Water permeability of polymerized vesicles Water permeabilities of both liposomes and proteoliposomes at different AqpZ-tolipid ratios were investigated using a stopped-flow spectrometer, and results were shown in Figure 3.5 Since the permeability of DOPC liposomes increases strikingly with temperature [115], all the permeability tests on stopped-flow were performed at 8°C to reduce the permeability noise derived from the lipid bilayer Without AqpZ incorporation (control), the permeability of cross-linked liposomes has been observed 55 about to be approximately 40% lower than that of the non-cross-linked liposomes It indicates that the formation of the polymethacrylate network in the hydrophobic phase of liposomes could resist water permeation across the lipid membrane Sizes of control liposomes with and without cross-linking were measured by DLS on the 1st and 2nd day of extrusion (see Table 3.1) The cross-linked liposomes show an almost unchanged polydispersity index (PDI) on the 2nd day, while the non-polymerized liposomes displayed an obvious increased PDI Since plain liposomes have the tendency to aggregate and fuse together, the cross-linked polymer mesh has stabilized the vesicle structure and prevented fusion of vesicles Figure 3.5 Water permeability of liposomes and proteoliposomes with different AqpZ-to-lipid weight ratios 56 Table 3.1 DLS results of control liposomes with and without UV cross-linking Sample Mean Diameter Polydispersity index (PDI) 1st day after extrusion 129.4 0.066 day after extrusion (with UV) 136.7 0.068 2nd day after extrusion (w/o UV) 149.5 0.132 2nd With AqpZ incorporation, the water permeability of non-cross-linked vesicles shows a significant improvement, from 29.4 µm s-1 of the control to 241.6 µm s-1 of the sample containing a protein-to-lipid weight ratio of 1:100 This result is lower than the findings by Borgnia et al in 1999 [37] in which they reconstituted AqpZ into liposomes of E coli total lipid extract and observed a water permeability of about 400 µm s-1 Apart from intrinsic differences in AqpZ, the lower permeability of our proteoliposomes could be owing to a lower AqpZ incorporation efficiency This may be caused by PEG brushes on the vesicle surface and/or hydrophobic-hydrophobic interactions between EGDMA and AqpZ The permeability of proteoliposomes was increased as the protein-to-lipid ratio was increased, but not proportionally This may be attributed to the fact that, at a ratio of 1:50 and 1:10, more detergent was added into the vesicle solution together with the AqpZ stock solution A large amount of detergent may cause solubilization of lipid membranes and thus result in a low AqpZ incorporation efficiency The water permeability of UV polymerized proteoliposomes was shown to be lower than the unpolymerized proteoliposomes Although the lipid membrane permeability was largely reduced due to UV polymerization as mentioned earlier, the incorporation of AqpZ has negated this small reduction in permeability Thus, we may conclude 57 that polymerization could compromise the functionality of AqpZ Formation of the polymer network may create an internal stress in the lipid bilayer, which also indirectly affects the protein structure However, these effects may be reduced if the AqpZ-to-lipid weight ratio approaches or beyond 1:10 Figure 3.6 FESEM images of the PAN membrane (a) before and (b) (c) after PDA coating, and (d) (e) with vesicle immobilization on the PDA coating A PDA layer with a thickness of about 100nm can be observed from the crosssection image of the PDA-PAN membrane in (c) Two immobilized vesicles were imaged at a higher magnification in (e) 3.3.4 Vesicle immobilized nanofiltration membranes Comparing with the blank PAN membrane (Figure 3.6(a)), both the surface pore size (Figure 3.6(b)) as well as the membrane permeability (Table 3.2) were reduced by the 100 nm PDA coating on the PAN surface (see Figure 3.6(c)) The cross-linked DOPC/DSPE-PEG-NH2 vesicles were then immobilized on the PDA surface For nanofiltration membrane fabrication, a slight vacuum was applied under the PDA- 58 PAN substrate to drive the vesicles to block the top surface pores, and at the same time these vesicles were more likely to bond with the PDA layer With this method, the vesicle solution on the membrane surface can be concentrated to a much higher concentration Finally, the immobilized layer was further cross-linked with glutaraldehyde Figure 3.6 (d) and (e) show the PDA surface covered with polymerized vesicles after hours of PWP tests at bar Interestingly, structure of many vesicles was still maintained showing that these vesicles were stable under the pressure Some fractured vesicles on the membrane surfaces displayed as hollow spheres with diameters of 100-300 nm The wall thickness of the cross-linked vesicles is estimated as less than 20 nm from the FESEM images For immobilization of vesicles without UV polymerization, very few vesicles were found on the membrane surface by FESEM after the PWP and salt rejection tests and the membrane cannot stand pressure higher than bar PWP results of the membrane before and after vesicle immobilization are shown in Table 3.2 After the immobilization of vesicles, the membrane flux drops dramatically indicating a high coverage of the surface pores by vesicles The salt rejection and permeate flux results for both NaCl and MgCl2 after vesicle immobilization are shown in Figure 3.7 59 Table 3.2 Pure water permeability (PWP) of PAN membrane before and after modification The standard error of the data is within 8% *The AqpZ-to-lipid weight ratio is used here Membrane PWP (LMHbar-1 ) Tested under bar Blank PAN (12%) 970 3hr-PDA-PAN 455 No AqpZ AqpZ:Lipid= 1:100* 3.8 AqpZ:Lipid= 1:50* 4.5 AqpZ:Lipid= 1:10* PDA-VesicleGlutaraldehyde 2.3 9.1 Figure 3.7 Salt rejection of vesicle immobilized PAN membranes with different AqpZ-to-lipid weight ratios (Testing pressure: bar Feed solution: 200ppm NaCl or 200ppm MgCl2.) Compared to the membrane without AqpZ incorporation, the membrane comprising the AqpZ-to-lipid weight ratio of 1:100 increases the PWP of 65% with enhanced NaCl and MgCl2 rejections of 66.2% and 88.1%, respectively Clearly, proteoliposomes incorporated with AqpZ possess high selectivity to ions The 60 vesicles without AqpZ behave as a dense selective layer providing a great resistance to permeation flow, while AqpZ opens up the flow channels as well as controls the rejection However, a trade-off between membrane flux and salt rejection was observed when AqpZ incorporation ratio increases The decreased salt rejection may be explained by defect formation in the polymerized networks due to a large amount of AqpZ embedded in proteoliposomes 3.4 Conclusion In this study, we have explored the possibility of fabricating a stable aquaporin reconstituted biomimetic membrane for nanofiltration We have shown that, with UV polymerization of EGDMA in the bilayer membrane, AqpZ can be still functionally reconstituted into the vesicles The membrane formed by immobilizing the designed vesicles on a porous membrane via amine-catechol adduct formation can withstand hydraulic pressure up to about bar as well as stirring shear The preliminary nanofiltration data demonstrate that, compared to the control, the membrane with AqpZ incorporation exhibits a higher pure water permeability as well as enhanced NaCl and MgCl2 rejections Obviously, AqpZ opens up the flow channels as well as controls the rejection Although there could be a few ways to improve the membrane performance, this work demonstrates that AqpZ incorporated proteoliposomes may potentially modify ultrafiltration membranes for nanofiltration Although in this work, immobilization of AqpZ incorporated proteoliposomes on microporous membrane was successfully demonstrated, both membrane flux and ion rejections of the biomimetic membrane were still below our expectations This may be attributed to a low usage of AqpZ channels in this design Since the vesicles are 61 directly immobilized on the microporous surface, the effective aquaporin for water transport is only limited to the ones on top of the pore area And because the pore size of the substrate membrane is only approximately 10-15 nm, only a very small fraction of AqpZ functions in the nanofiltration process In the following works, solutions to this problem were introduced by embedding the proteoliposomes into a relatively impermeable membrane matrix ... 2. 2 Materials and methods 2. 2.1 Materials Nickel-chelating lipids, 1 ,2- di-(9Z-octadecenoyl)-sn-glycero-3-(N-(5-amino-1- carboxypentyl)iminodiacetic acid)succinyl with Nickle (DOGSNTA) and 1,2dipalmitoyl-sn-glycero-3-phosphocholine... images in Figure 2. 2 Without the addition of BioBeads, the DPPC bilayer loses the original compact structure (Figure 2. 2(a)) and forms a “fluid-like” defective structure (Figure 2. 2(b)), while with... water (L h-1), ∆P is the transmembrane pressure difference (bar) and A is the membrane surface area (m2) Rejections of NaCl and 51 MgCl2 solutions (20 0ppm in ultrapure water) were tested with a surface