62 Chapter Aquaporin-embedded Mixed-Matrix Membrane: A Layer-by-Layer Self-assembly Approach 4.1 Introduction Among different water purification techniques, reverse osmosis and nanofiltration become more and more popular for drinking water production because of their effectiveness in removing low molecular weight impurities such as small organic compounds and ions [116] However, reverse osmosis and nanofiltration are still energy-intensive processes, and it is crucial to develop high-performance membranes with new materials AQPs in biological membranes are water channel proteins that are precisely engineered by nature [36, 106] The exceptional selectivity and permeability that AQP demonstrates towards water molecules make it a potential candidate in designing high-performance membranes for water purification Among the AQP family, AqpZ has attracted particular attention in biomimetic membrane research [88, 106, 117, 118] The functional reconstitution of AqpZ in both lipid bilayer and block copolymer membranes has been demonstrated previously [37, 38] Kumar et al have reported that the water permeability of an AqpZ incorporated biomimetic membrane could possibly reach 80 times higher than current commercial reverse osmosis membranes [38] Motivated by this finding, several attempts have been made to develop AqpZ embedded biomimetic membranes for different applications such as forward osmosis[86], reverse osmosis [88], or nanofiltration [87, 119] However, even through some of previous works have demonstrated water transport ability of AqpZ in their membranes, the membrane fabrication in a larger scale remains a challenge, especially when the membrane stability and integrity have to be maintained for long-term operations 63 In this work, a practical design of the AqpZ embedded mixed-matrix membrane (MMM) was introduced using the layer-by-layer (LbL) adsorption approach The LbL matrix has offered a stable and compatible environment for AqpZ-reconstituted vesicles and this design may become one of the potential solutions for fabricating the AqpZ-assisted water purification membranes The LbL film is formed by alternatively depositing polycations and polyanions onto a charged substrate It has been studied in a wide variety of research topics As a versatile and convenient strategy, previous research works have demonstrated the functionalization of LbL films via the incorporation of various compounds of interest such as DNA [120], proteins and enzymes [121, 122], nanoparticles [123], and even liposomes [124, 125] The embedding of liposomes into the polyelectrolyte multilayers has been investigated for the development of drug release or enzymatic nano-reactor systems [124-126] Polyelectrolyte encapsulated liposomes have demonstrated better chemical [127] and mechanical stabilities [128] than intact liposomes Gentle polyelectrolytes such as poly-L-lysine (PLL) have to be used for liposome encapsulation because strong polyelectrolytes could disrupt the lipid membranes during the adsorption process Previous works have shown that PLL-covered liposomes are able to be absorbed onto various polyanion films [126] In our current work, the AqpZ reconstituted proteoliposomes are first stabilized with PLL and then embedded within the LbL film that is formed on a porous membrane for nanofiltration studies The adsorption of PLL onto the proteoliposome surface may not damage the incorporated protein because PLL is a biocompatible material composed of amino acids [129] 64 To fabricate the LbL film, polyallylamine hydrochloride (PAH) was used to form the polycation layer, while a blend of polyacrylic acid (PAA) and polystyrene sulfonate (PSS) was used to form the polyanion layer Since PAA is a weak polyanion but has good hydrophilicity, while PSS is a strong but rather hydrophobic polyanion, the blended PSS/PAA system provides both hydrophilicity and relatively strong interactions with polycation layer [130] It has also been reported that LbL films formed by a combination of PAH-PSS/PAA have a lower surface roughness [131] and a better ion rejection [132] than films formed by a single polyanion (PAA or PSS) paired with PAH The positively charged PLL-covered liposomes will be encapsulated within the LbL film to form a nano-composite MMM, as shown in Figure 4.1 The polyelectrolyte layers act as a continuous matrix phase and the AqpZ incorporated liposomes function as the highly permeable dispersed phase The aim in this paper is to explore the feasibility of devising the AqpZ-embedded MMM for pressure-driven water purification, and also to demonstrate the functionality of AqpZ in the MMM Hydrolyzed PAN membrane Poly-L-lysine encapsulated liposome with water channel proteins Figure 4.1 The schematic presentation of the formation procedures for the liposome-embedded LbL membrane 65 4.2 Materials and methods 4.2.1 Materials 1-palmitoyl-2-oleoyl-sn-gly-cero-3-phosphocholin (POPC), 1-palmitoyl-2-oleoyl-snglycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG), cholesterol were purchased from Avanti Polar Lipids Hydrochloric acid (HCl), sodium chloride (NaCl), magnesium chloride (MgCl2), sodium hydroxide (NaOH), n-methyl-2pyrrolidone (NMP), PLL, PAA, PSS, PAH, and glutathione (C10H17N3O6S, MW 307.33) were products of Sigma-Aldrich (USA) Bio-Beads SM-2 absorbents and tris(hydroxymethyl)aminomethane (Tris) were purchased from BIO-RAD (USA) Polyacrylonitrile (PAN) for substrate preparation was obtained from Tong-Hua Synthesis Fiber Co Ltd (Taiwan) Grade V1 mica was purchased from SPI Supplies (USA) Ultrapure water was produced by the Millipore Reference A+ system (Merck Millipore, USA) A 10 mM Tris buffer at pH=7.5 with 15 mM NaCl was used throughout this study 4.2.2 Vesicle preparation and characterization POPC and POPG were mixed for vesicle formation at certain molar ratios together with 5% (w/w) cholesterol (Chol) A multilamellar vesicle suspension in a Tris 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, UK) that had a pore size of 100 nm For AqpZ reconstitution experiments, an AqpZ stock solution was added during the film rehydration step and the mixture was agitated for at least hours BioBeads were then added into the mixture stepwise to remove the detergent completely The suspension was protected with high purity argon throughout the experiment 66 The intact POPC/POPG/Chol liposomes were then stabilized with PLL according to the following procedures A mg ml-1 liposome solution was added at equal volumes to a 0.5 mg ml-1 PLL solution in Tris buffer dropwise, while the PLL solution was stirred at a speed of 950 rpm The mixing process was completed within minutes Lipo8, lipo15 and lipo30 refer to samples containing 8, 15 and 30 percent of POPG in the liposomes, respectively Similarly, PLL-lipo8, PLL-lipo15 and PLL-lipo30 represent PLL-covered liposomes with their respective POPG content A Zetasizer Nano ZS instrument (Malvern, UK) was employed to characterize the vesicle size distribution and zeta potential Field emission transmission electron microscopy (FETEM: JEOL, JEM-2100F, Japan) was used to image POPC/POPG/Chol liposomes before and after PLL adsorption Before imaging, the liposome solution were diluted to 0.5 mg ml-1 with Tris buffer and dropped on ultrathin carbon film coated copper grids for 15 and then rinsed with ultrapure water dropwise The samples were air dried for 30 before the FETEM imaging 4.2.3 Vesicle permeability measurement using stopped-flow Water permeabilities of both the intact liposomes and the PLL-stabilized liposomes at different AqpZ-to-lipid ratios were investigated using a stopped-flow spectrometer (Applied Photophysics, Chirascan, UK) By rapidly mixing the liposome solution with a hypertonic buffer solution containing 0.6 mol L-1 sucrose, water would diffuse from the vesicles into the buffer, causing the vesicles to experience a sudden shrinkage To improve the signal to noise ratio, all the experiments were conducted at 67 a temperature of 8°C Data have been fitted to Equation to estimate the rate constant k The final osmotic permeability (Pf) of the vesicles was calculated by Equation 4.2.4 Liposome-embedded LbL on a mica surface Three sets of PAH-PAA/PSS were prepared on newly cleaved mica surfaces by depositing solutions containing g L-1 of PAH and g L-1 of PSS/PAA (mixed at a weight ratio of 1:1) alternately onto the mica surface and rinsing with pure water after each deposition step Each deposition step lasted for 15 PLL-covered liposomes containing 15% or 30% POPG were then deposited on top of the third PSS/PAA layer and incubated for hours After the incubation, the mica was rinsed with buffer to remove unbound vesicles and then covered with one more layer of PSS/PAA The films were imaged by an AFM that was operated in an acoustic alternating current mode The samples were also scanned by OLYMPUS cantilevers (OMCLTR400PSA, resonance frequency of 11 kHz and a typical force constant of 0.02 N m1 ) in aqueous solutions (prepared with Milli-Q water) at room temperature (23 ± 1°C) 4.2.5 Liposome-embedded LbL membranes for nanofiltration Flat sheet PAN substrates were prepared by casting a 12 wt% PAN/NMP solution directly on glass plates with a 150 µm casting knife The membranes were then quickly immersed in a water bath to induce phase inversion and then soaked in deionized water overnight to remove all traces of NMP The PAN membranes were later hydrolyzed with a solution containing M NaOH for 1.5 hours at 50°C to generate negative charges on the membrane surface After hydrolysis, the membrane was washed with excess volumes of ultrapure water and used within one week 68 To prepare the nanofiltration membrane, a PAH-PSS/PAA bilayer was first deposited onto the surface of the hydrolyzed PAN for 15 The membrane was washed with ultrapure water for after each layer was deposited The PLL-liposome solution was incubated on top of the PSS/PAA layer for hours and gently spray-washed with ultrapure water Finally, one more layer of PSS/PAA was deposited on top of the PLL-liposomes for stabilizing the liposomes A membrane area of 78.5mm2 was used for nanofiltration 4.2.6 Nanofiltration studies The nanofiltration tests were conducted using dead-end permeation cells The measurements for the pure water permeability (PWP) were conducted at 23 ± 1°C in terms of L m-2 h-1 bar-1, with the selective layers facing the feed at a trans-membrane pressure of bar The PWP was calculated from Equation The rejections of 200ppm MgCl2 and glutathione solutions in ultrapure water were tested using a surface mixing speed of 700 rpm at a trans-membrane pressure of bar The salt rejection was calculated using the Equation Each reported data was an average of at least three different samples The solute permeability (B) was estimated using the following equation [133]: ! = ! ∆! − ∆! 1−! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(7) ! where A is the intrinsic water permeability or PWP, ΔP is the trans-membrane hydraulic pressure and Δπ is the osmotic pressure from the feed solution On the basis of the dissociation constants of glutathione, i.e., the pKa values, the fraction of glutathione present in different ionization states at different pH values can be expressed by the following the Henderson-Hasselbalch equation shown below 69 [134] The concentration of glutathione was measured by a total organic carbon analyzer (Shimazu, TOC ASI-5000A, Japan) pH = p!! + log !( [!"#$#%!!""#$%&'] )!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(8) [!"#$#%!!"#"$] 4.3 Results and discussion 4.3.1 PLL adsorption on liposomes To study the vesicle properties at different POPG content, the mean vesicle diameters and the zeta potentials of both the intact and the PLL-stabilized liposomes were measured using a Zetasizer apparatus, as shown in Table 4.1 For the intensityweighted mean diameters of all the samples, the polydispersity indexes are always less than 0.15, indicating that the particle sizes follows a narrow mono-dispersed distribution Upon mixing the intact liposomes with PLL, the mean vesicle sizes increase because PLL is adsorbed onto the negatively charged lipid bilayer The zeta potential becomes more negative as the content of POPG is increased in the liposome because of the accumulation of a higher charge density as POPG is a negatively charged lipid Consequently, as the POPG content is increased, more PLL molecules could be adsorbed onto the liposome surface, producing PLL-covered liposomes with a higher zeta potential However, despite the fact that more PLL molecules are adsorbed onto the liposomes that possess higher POPG content, a stronger electrostatic interaction induces a more extended and a more compact molecular structure of PLL Thus, the thickness of the PLL layer is reduced with an increasing amount of POPG in the liposomes POPC/POPG/Chol liposomes before and after PLL adsorption were imaged with FETEM and shown in Figure 4.2 In accordance with the dynamic light scattering results, bolder vesicle walls can be observed from the PLL-liposome images because of the PLL adhesion outside the lipid bilayer 70 200#nm# 100#nm# 200#nm# 200#nm# (a) (b) Figure 4.2 FETEM images of (a) intact liposomes, and (b) PLL-covered liposomes The liposomes content 15% POPG Table 4.1 The zeta potential and the intensity-weighted mean hydrodynamic diameter of the intact liposomes and the PLL-covered liposomes The thickness of the adsorbed PLL layer was calculated from the diameters obtained Three different liposome samples were used to obtain the mean and the standard deviation PLL thickness** (nm) Sample Mean Diameter* (nm) Zeta potential* (mV) Lipo8 136.6±5.4 -41±4 Lipo15 139.7±2.9 -48±2 Lipo30 141.7±4.0 -61±4 PLL-lipo8 158.5±5.1 32±2 11±1.6 PLL-lipo15 154.6±1.9 46±3 7.5±0.5 PLL-lipo30 153.4±3.6 58±2 5.8±1.8 * Three different liposome samples were used to obtain the mean and the standard deviation ** The thickness of the adsorbed PLL layer was calculated from the diameters obtained 71 4.3.2 Water permeability measurement by stopped-flow Stopped-flow light scattering was used to study the water permeability of POPC/POPG/Chol vesicles at different AqpZ incorporation ratios The measurements were conducted at 8°C to reduce the noise level Only the results from the liposomes containing 30% POPG are presented here because the POPG content did not influence the function of AqpZ An increase in the light scattering signal corresponds to a reduction in the vesicle size after hypertonic osmotic shock Moreover, the increased rate that is represented by the rate constant k is directly related to the osmotic permeability of the vesicles (Equation 4) The normalized signals of the intact liposomes with different AqpZ-to-lipid ratios are compared in Figure 4.3(a) With the incorporation of AqpZ, a rapid increment in light scattering signal can be observed in the first 40 ms However, the control liposomes that were prepared without AqpZ took approximately 1.5 seconds to reach steady state, which was a much slower response in comparison with the AqpZ-incorporated vesicles The osmotic permeabilities of the intact liposomes and the PLL-covered liposomes are shown in Figure 4.3(b) From the stopped-flow results, the PLL adsorption does not appear to have much effect on lipid membrane permeability or AqpZ functionality As the AqpZ-to-lipid ratio was increased from to 1:50, the permeability increased approximately linearly from 13 µm/s to 853 µm/s However, a further increase in the vesicle permeability was not observed at higher AqpZ incorporation ratios This finding is similar to the results that were presented in the previous work [37], and the possible reason could be the interference of a large amount of detergent in the AqpZ incorporation process [38] 72 Normalized light scattering (A.U.) 3.5 (a) 1:25 1:50 2.5 1:100 1:200 1.5 Control 0.5 0 0.02 0.04 0.06 Time (s) 0.5 1.5 (b) 1000 800 Pf (µm/s) 600 400 Intact liposome PLL-covered liposome 200 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 AQPZ-to-lipid weight ratio Figure 4.3 The liposome permeability measured by stopped-flow light scattering (a) The kinetics of water permeability at different AQPZ-to-lipid ratio (a representative plot is shown for each sample) Each plot was normalized to fit between and unity An increase in light scattering signal represents a reduction in vesicle volume due to water efflux (b) The comparison of the calculated permeability Pf of intact liposomes and PLL-covered liposomes at different AQPZ-to-lipid weight ratios 4.3.3 Embedding PLL-covered liposome in LbL films Because PLL adsorption does not affect the AqpZ performance, the LbL film embedded with AqpZ incorporated liposomes are constructed for the subsequent studies The selection of the polyanion is critical for the uniform distribution of liposomes in the LbL film [126] To study the PLL-covered liposome interaction with different polyanions, 0.5 mL PLL-stabilized liposome (PLL-lipo15 and PLL-lipo30) suspension with a concentration of 0.5 mg/mL were added into 0.5 mg/mL polyanion 73 solution of the same volume Then, the mixture was vortexed for 30 s to ensure the uniform mixing As shown in Figure 4.4, the vesicle size is reduced back to the intact liposome size after mixing the PSS solution with the PLL-liposome solution, indicating that PLL desorbs from the liposome surface By blending a weaker polyanion (PAA) with PSS at 50/50, the overall ionic strength is reduced to the extent that the polyanion is adsorbed outside the PLL-liposome and the vesicle diameter is increased after the PSS/PAA solution is mixed with the PLL-liposome solution This observation suggests that the PSS/PAA blend of polyanions works better than a pure PSS polyanion for maintaining the stability of the PLL-covered liposomes Therefore, in our study, a PSS/PAA blend was adopted to form the polyanionic layers in the LbL film 200 Lipo15 Mean diameter (nm) 190 Lipo30 180 170 160 150 140 130 PS S d w ith PS w bl S/ PA A ith en m so po -li PL L Li po so m e e 120 Figure 4.4 Intensity-weighted mean diameters of intact liposomes, PLLliposomes and PLL-liposome mixed with different polyanions To evaluate the adsorption ability of PLL-liposomes onto the LbL films, PLL-lipo15 and PLL-lipo30 solutions were deposited on the (PAH-PSS/PAA)3 films that were 74 formed on mica surfaces and followed by one more layer of PSS/PAA Using AFM, the embedded liposomes in the LbL film were observed clearly, as shown in Figure 4.5 Liposomes containing greater amounts of charged lipid, after covered with PLL, have stronger interaction strengths with the PSS/PAA layer From the AFM images, PLL-lipo30 interacts more actively with the negatively charged PSS/PAA layer than PLL-lipo15, as shown by the larger amount of adsorbed vesicles Thus, the surface coverage of the entrapped liposomes can be increased by increasing the content of charged lipids in the liposomes (a) (PAH-PSS/PAA)3-PLL-lipo15PSS/PAA (b) (PAH-PSS/PAA)3-PLL-lipo30PSS/PAA Figure 4.5 The AFM images of (a) the PLL-lipo15 and (b) the PLL-lipo30 embedded LbL membranes on the mica substrate The dimensions of both images are ì6 àm 75 (a) (b) 500nm 500nm (d) (c) (e) Embedded liposomes 500nm 100nm 500nm Figure 4.6 The FESEM images of (a) blank hydrolyzed PAN membrane, (b) a control LbL film without embedding any liposomes, (c) (d) a liposome-embedded LbL film before filtration tests, (e) a liposome-embedded LbL film after filtration tests The slanted view in (d) demonstrates that the liposomes are partially embedded in the polyelectrolyte matrix 4.3.4 Liposome embedded membrane for nanofiltration As the hydrolysis of PAN may generate negative charges on the membrane surface, the positively charged PAH could be directly adsorbed onto the hydrolyzed PAN membrane surface [135] The water permeability of the hydrolyzed PAN membrane is approximately 67 L m-2 h-1bar-1 With the deposition of a PAH-PSS/PAA bilayer, the permeability of the membrane is reduced to 8.6 L m-2 h-1 bar-1 It is therefore obvious that the polyelectrolytes formed stable and relatively dense layers on the surface of the hydrolyzed PAN membrane The control LbL film was prepared by further depositing a PLL-PSS/PAA bilayer on the top of the PAH-PSS/PAA bilayer The surface morphology of the control LbL membrane is observed to be smooth and uniform from FESEM imaging, as shown in Figure 4.6(b) The surface pores in the blank hydrolyzed PAN membrane (Figure 4.6(a)) are completely covered by this dense selective layer The intrinsic water permeability of the control LbL membrane 76 is approximately 3.8 L m-2 h-1 bar-1 For liposome-embedded LbL films, scattered vesicular structures are clearly shown in FESEM images before and after the nanofiltration experiments (Figure 4.6(c-e)) The diameters of these structures are approximately 100-200 nm, which correspond to the size of the liposomes that are produced in our experiments Figure 4.6(e) demonstrates that these PLL-covered liposomes could survive in the nanofiltration test, which was performed under a hydraulic pressure of bar as well as surface perturbance PLL-liposomes with different POPG content were embedded into the LbL film for nanofiltration experiments, as shown in Figure 4.7 and Table 4.2 For the AqpZ-embedded membrane, a protein-to-lipid ratio of 1:100 was applied For the membranes that are embedded with liposomes containing no AqpZ, the water permeabilities are lower than the control LbL films The membrane permeability tends to decrease at a higher amount of POPG in the liposomes In contrast, with the incorporation of AqpZ, the membrane permeabilities are higher than that of the control LbL film, where an increased POPG content in liposomes increases both the membrane permeability and salt rejection The AqpZ incorporation significantly enhances the water permeability (A) without increasing the solute permeability (B) as shown in Table 4.2, especially for the PLL-lipo15 and the PLL-lipo30 embedded membranes The results have clearly demonstrated the function of AqpZ in the MMM, which exhibit a high permeability for water molecules only 77 Rejection-w/o AQPZ Rejection-w AQPZ Permeability-w/o AQPZ Permeability-w AQPZ 100% 98% 96% 94% 92% 90% 88% Salt Rejection Permeability (Lm-2h-1bar-1) 86% PLL-lipo8 PLL-lipo15 PLL-lipo30 Figure 4.7 The water permeability (marker) and the MgCl2 rejection (column) of the LbL film embedded with AqpZ-reconstituted vesicles or AqpZ-free vesicles The FESEM images demonstrate an increase in liposome adsorption as POPG content is increased Table 4.2 The intrinsic water permeability and the MgCl2 permeability of the blank substrate and the substrate with LbL films.* Intrinsic water permeability Solute permeability A (L m-2 h-1 bar-1) B (L m-2 h-1) Hydrolyzed PAN (Substrate) 67.4±5.6 - PAH-PSS/PAA 8.61±0.12 6.58±1.71 PAH-PSS/PAA-PLL-PSS/PAA (Control LbL) 3.82±0.06 2.02±0.19 PAH-PSS/PAA-PLL-lipo8-PSS/PAA 3.28±0.24 1.56±0.03 PAH-PSS/PAA-PLL-lipo15-PSS/PAA 2.4±0.57 0.97±0.26 PAH-PSS/PAA-PLL-lipo30-PSS/PAA 2.45±0.13 0.81±0.09 PAH-PSS/PAA-PLL-lipo8-PSS/PAA 4.77±0.45 2.00±0.45 PAH-PSS/PAAS-PLL-lipo15-PSS/PAA 5.33±0.10 0.98±0.24 PAH-PSS/PAA-PLL-lipo30-PSS/PAA 6.13±0.22 1.16±0.18 Membrane No AQPZ AQPZ 78 *Feed: 200ppm MgCl2; three samples were used to obtain the mean and the standard deviation Similar as the findings from the AFM images (Figure 4.5), in the resultant nanofiltration membrane, more liposomes can be embedded into the LbL film at higher POPG content, as shown in the FESEM images in Figure 4.7 The membrane permeability is highly correlated with the liposome coverage in the LbL film because AqpZ-incorporated liposomes are highly permeable particles By increasing the POPG content, the liposome coverage is enhanced, thus enhances the membrane permeability as well Without AqpZ, the liposomes are almost impermeable such that membranes embedded with more liposomes demonstrate lower permeabilities Nevertheless, as we increased the AqpZ-to-lipid ratio in the embedded liposomes, the membrane permeability was not substantially enhanced (data not shown) because the designed membrane suffered from a low liposome-embedding efficiency Even for the LbL film that contained PLL-lipo30, the vesicular fraction is only approximately 20%, as estimated from the FESEM images The future work will be focused on improving the vesicle encapsulation efficiency by eliminating the unbounded PLL in the PLL-liposome solution or by other methods 79 Rejection of glutathione 0.8 +1 -1 0.6 -3 -2 0.4 0.2 0 pH 10 12 Figure 4.8 The rejection of glutathione at various solution pH values (red solid line) The membrane used was an LbL film that was embedded with PLL-lipo30 (AqpZ-to-lipid ratio was 1:100) Glutathione can be ionized into five different states bychanging the solution pH The fraction of each state is indicated in the figure as dotted curves pKa1=2.12, pKa2=3.59, pKa3=8.75, pKa4=9.65 This newly-developed biomimetic membrane could be potentially applied to the nanofiltration process for the recovery or the concentration of small molecules from aqueous solutions The membrane with AqpZ-incorporated PLL-lipo30 was tested for its glutathione rejection at various pH levels (2.8, 4, 7, 9) Glutathione is the major thiol compound in our body that works as an antioxidant and detoxifier [136, 137] It has been widely applied as a supplement or a therapeutic drug for the treatment of cancer, HIV, Parkinson’s disease, and Alzheimer’s disease [138, 139] This tripeptide molecule has multiple ionization states as shown in the dotted curves in Figure 4.8, because it contains amino, thiol and carboxyl groups As shown in Figure 4.8, the rejection of glutathione by the membrane is 86% when the solution pH is at the isoelectric point of glutathione (2.12-3.59), where glutathione is electrically neutral At this pH range, the size exclusion mechanism controls the membrane rejection 80 properties As the solution pH is increased, the glutathione becomes negatively charged and the membrane rejection rate increases to 93%, indicating that electrostatic repulsion plays a functional role in the nanofiltration process However, although a further increase in pH (up to 9) induces a higher ionic strength, the rejection of glutathione remains unchanged Thus, the size exclusion mechanism is more significant than the electrostatic exclusion effect in this LbL biomimetic membrane 4.4 Conclusion In this work, a biomimetic MMM embedded with AqpZ was designed and fabricated using the polyelectrolyte LbL self-assembly approach The membrane incorporated with AqpZ has shown enhanced water permeability and salt rejection in comparison with the control membrane Several advantages of this design can be identified: (1) the relatively impermeable matrix eliminates or reduces the defects in the interliposome area so that water molecules are more easily transported through the AqpZ channels; (2) the AqpZ-incorporated liposomes behave as a highly permeable dispersed phase that increases the overall permeability of the membrane; (3) the properties of both continuous and dispersed phases can be effectively controlled by properly selecting the polyelectrolytes and the lipids, respectively The LbL approach makes the production of AqpZ-embedded biomimetic membranes more efficient and also opens up the possibility of large-scale fabrication  ... Tris buffer and dropped on ultrathin carbon film coated copper grids for 15 and then rinsed with ultrapure water dropwise The samples were air dried for 30 before the FETEM imaging 4.2 .3 Vesicle... PAH-PSS/PAAS-PLL-lipo15-PSS/PAA 5 .33 ±0.10 0.98±0.24 PAH-PSS/PAA-PLL-lipo30-PSS/PAA 6. 13? ?0.22 1.16±0.18 Membrane No AQPZ AQPZ 78 *Feed: 200ppm MgCl2; three samples were used to obtain the mean and the standard deviation... antioxidant and detoxifier [ 136 , 137 ] It has been widely applied as a supplement or a therapeutic drug for the treatment of cancer, HIV, Parkinson’s disease, and Alzheimer’s disease [ 138 , 139 ] This