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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers
Reversibly attached phospholipid bilayer functionalized membrane pores Anju Kumari, Lavie Rekhi, and Saurav Datta Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03404 • Publication Date (Web): 05 Nov 2018 Downloaded from http://pubs.acs.org on November 6, 2018
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Reversibly attached phospholipid bilayer functionalized membrane pores Anju Kumari, Lavie Rekhi, Saurav Datta* Department of Biotechnology, Indian Institute of Technology Roorkee
* Corresponding author:
Saurav Datta Department of Biotechnology Indian Institute of Technology Roorkee Roorkee-247667, Uttarakhand, India Email:
[email protected] Tel: +91-1332-284795
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Abstract: We report development of reversibly attached phospholipid bilayer (PLB) functionalized membrane pores that enabled reusability of the membrane matrix as well as the phospholipid. The functionalized architecture was constructed based on electrostatic interactions, which facilitate reversible attachment-detachment sequence of the functional moieties within membrane pores. To demonstrate potential application, an enzyme, glucose oxidase (GOx), was electrostatically immobilized within the phospholipid bilayer functionalized membrane and enzymatic catalysis was conducted under convective flow mode. GOx-immobilized membrane demonstrated satisfactory activity and stability. Convective flow of substrate solution resulted in significantly higher activity than diffusive flow. Then, the enzyme was detached keeping the functional PLB backbone intact. Detachment of the enzyme without affecting the functional activity of PLB backbone permits attachment of fresh enzyme. In addition, reusability of the phospholipids is also of great importance as they have wide range of applications, but their usage is limited by higher cost. We have demonstrated detachment of the PLB from the membrane using a simple technique. Characterization of the detached phospholipid confirmed retention of the original structural and functional properties as exhibited before attachment. To the best of our knowledge, this is the first study on reversible phospholipid bilayer formation within membrane pores and demonstration of a detachment technique, while maintaining the structural and functional properties of the phospholipid.
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Introduction: Deposition of lipid bilayer on a solid support, known as supported lipid bilayer (SLB), has attracted wide attention from scientific community by virtue of their stability and fluidity to mimic biological cell membranes 1. Examples include development of drugs targeting cell surface, monoclonal antibody development
4–6
2,3
disease cell recognition by using nanoparticles 7, designing
biocompatible interfaces for enzyme immobilization, biosensor development, lipid-protein interaction,
8,9
etc. Formation of SLB is a spontaneous process, which occurs essentially by two
techniques – Langmuir–Blodgett (LB) and Vesicle-Fusion (VF). The latter one, which involves fusion of small unilamellar vesicles (SUVs), has the advantages of versatility and simplicity over LB technique 10. SUVs are spherical, enclosed structure of lipid bilayer. The mechanism of SLB formation from SUVs via VF technique is thoroughly described somewhere else 11. The essence of the mechanism is illustrated in Figure 1. SUVs from solution phase, when spread onto a solid surface, approach towards each other, fuse and flatten to form liposomal structure. Then, the liposomal structure ruptures on both ends resulting into two lipid bilayers, one above the other. Two lipid bilayers slide on opposite directions to expand and eventually lead to bilayer formation. Single SUV could also lead to SLB formation following steps (iii) to (v) of the above mentioned mechanism.
SUVs in solution
(i)
(iii)
(ii)
(iv)
(v)
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Figure 1. Mechanism of supported lipid bilayer (SLB) formation on a solid surface following Vesicle-Fusion technique using small unilamellar vesicles (SUVs). (i) Approach, (ii) Fusion, (iii) Flattening, (iv) Expansion/sliding, and (v) SLB In SLB, typically, a thin water layer of 10-20 Å is trapped between the lipid bilayer and the solid support 12. This thin water layer is unable to restrict the unproductive interactions between the solid support and the SLB that hinder lateral mobility of phospholipid molecules
13–15.
Therefore, a cushion of water compatible polymer has been included between the underlying solid support and the SLB. Hydrated cushion serves as a lubricant and allows the desired structural mobility of the SLB
16.
Polymer cushion also impersonates cytoskeletal support found in
mammalian cells 17. Various materials have been used as cushion between SLB and the underlying support 9,18– 20.
Zhang et al. (2000) incorporated cationic poly(diallyldimethylammonium chloride) (PDDA)
layer between bilayer of anionic lipid 1-stearoyl-2-oleoylphosphatidylserine (SOPS) and solid gold surface. Electrostatic interaction between cationic PDDA and negatively charged SOPS formed a single lipid bilayer with the desired mobility at room temperature 21. Polyethyleneimine (PEI) as a polymer cushion has been implemented widely for the fabrication of SLB
12,22,23,
but
membranes as support materials for polymer-cushioned SLB are comparatively new. Polymeric membranes offer many advantages to bioprocess industries. Being porous, they offer high surface area per unit volume (higher loading capacity), promote pressure-driven convective flow (faster processing), and provide easy and linear scale-up opportunities (favorable commercialization potential)
24–27.
Despite several studies on inclusion of polymer cushion between SLB and solid
support, the reusability of the SLB functionalized solid support by reversible attachment of lipid bilayer is not explored, yet. Earlier, we have reported the formation of SLB within functionalized nylon membrane pores and demonstrated its efficacy in enzymatic catalysis using glucose oxidase (GOx) as a model enzyme 24. Herein, for the first time, we investigate the reversibility of the electrostatically attached SLB within polymeric membrane pores. We designate the electrostatically immobilized phospholipid bilayer as PLB. Reusability of the detached phospholipid for the formation of PLB was demonstrated with the help of Transmission Electron Microscope (TEM) as well as by reattaching it within functionalized membrane pores. Further, to demonstrate potential application 4 ACS Paragon Plus Environment
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of the PLB functionalized membrane, glucose oxidase (GOx) enzyme was electrostatically immobilized and enzymatic catalysis was conducted. Then, the enzyme was detached keeping the functional PLB backbone intact. Overall, we present a reversibly attached phospholipid bilayer (PLB) functionalized membrane that enables reusability of the membrane matrix as well as the phospholipid bilayer. 2. Materials and Methods: 2.1. Materials: Ultipor® Nylon 66 membrane discs (membrane diameter of 25 mm, pore diameter of 0.2 μm, average thickness of 160 μm) used for all the experiments, were purchased from Pall Corporation (Product No. NR025100). Nylon membrane was selected as the base membrane due to its hydrophilicity and availability of functional groups for further modification. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was purchased from Avanti Polar Lipids, USA (Product No. 850345P). Glucose oxidase enzyme from Aspergillus niger (GOx, Product No. RM7064, MW 160000) and Bradford reagent (Product No. ML106-500ML) were purchased from HiMedia Laboratories, India. β-D(+)-glucose (Product No. 20117) and sodium chloride (Product No. 20241) were purchased from s d fine-chem Limited, India. Epichlorohydrin (ECH, Product No. 027706) was purchased from Central Drug House (P) Ltd., India. Polyethyleneimine (PEI, Product No. 408727, MW ~25,000) and 15 % solution of titanium oxysulfate in dilute H2SO4 (Product No. 495379) were purchased from Aldrich Chemical Co., USA. All the experiments were conducted with ultrapure water. 2.2. Experimental Methods: All the experiments were performed in triplicate and the observed variations in experimental results are represented as standard deviation. 2.2.1. Membrane experiments: A membrane holder (25 mm disc diameter, dead-end type) was used to conduct experiments under convective mode of flow. Inlet and outlet of the membrane holder were connected to the feed tank and sample collection tank, respectively. To maintain the desired flow rate, a peristaltic pump was used. For the activity analysis of immobilized GOx, continuous flow was maintained. 2.2.2. Preparation of SUVs from DMPC: A technique described by Jass et al.
11
was
followed for formation of SUVs from DMPC. DMPC was dissolved in chloroform (1 mg/mL) and the mixture was kept overnight in vacuum oven at 40 0C to evaporate extra solvent and form a thin film of DMPC. The resultant lipid film was then reconstituted with 0.1 M NaCl solution to get a final concentration of 0.7 mg/mL. The solution was then vortexed and sonicated (probe sonicator, 5 ACS Paragon Plus Environment
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50 % duty cycle) for 20 min to form SUVs in solution. After sonication, the SUV-containing solution was centrifuged at 10,000 rpm for 15 min to remove impurities, and SUVs were collected in the supernatant. The supernatant containing SUVs was passed through PVDF syringe filter and dynamic light scattering (Zetasizer Nano ZS90) was conducted to ensure the desired size was Tm). A flow rate of 2 mL/min was maintained during the whole process. Functionalization of membrane pores was accomplished by immobilization of polymer cushion supported lipid bilayer as described below and depicted in Figure 2. First, the primary amine groups of nylon membrane were activated by recirculating 50 mL of 0.1 N HCl for 45 min. This was followed by recirculation of epichlorohydrin solution (50 ml of 1 M ECH in 0.5 M NaOH solution at 50 0C temperature) through the membrane for 45 min. Due to lower steric hindrance compare to the epoxide-carbons, primarily the chloride-carbon of ECH reacts with the amine groups of nylon membrane under alkaline condition, thereby introducing epoxide rings within the membrane pores for further functionalization
30-31.
These epoxide rings
were allowed to react with the primary amine groups of PEI (by permeating 50 mL of 0.2 mM PEI solution at pH 9 for 45 min) resulting into the formation of covalently attached polymer cushion of PEI. The polymer cushion of PEI within the membrane pores contained multiple secondary amine groups, which were further utilized for lipid bilayer formation by recirculating 5 mL SUV solution of DMPC for 45 min. Electrostatic interaction between the positively charged secondary amine groups of PEI and the negatively charged phosphate groups of the bottom layer of DMPC provided the required stability for the formation of the polymer cushion supported PLB within membrane pores. The upper layer of zwitterionic DMPC remained available for further attachment of functional groups. Covalently attached polymer cushion increased the space between the PLB and the membrane pore surface, thereby, reducing the friction and helping to maintain the
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structural integrity and desired fluidity of the attached PLB. Moreover, the functionalized moieties of PEI chains provided an extended platform to PLB with significantly higher pore coverage. 2.2.4. Immobilization of enzymes within biomimetic membrane: To immobilize GOx within the biomimetic membrane, 10 mL of the enzyme solution (200 µg/mL) was recirculated for 45 min through membrane pores at pH 6.5. Since the isoelectric point of GOx is around 4.2, at an operating pH of 6.5 the negatively charged GOx molecules electrostatically interacted with the positively charged amine groups of PLB (Figure 2). Any unbound enzyme was removed by washing with 25 mL of ultrapure water. Membranes containing immobilized enzymes were stored at 4 °C. 2.2.5. Activity analysis of the immobilized enzyme: PLB-functionalized GOximmobilized membrane was subjected to convective flow of the substrate (oxygen saturated glucose) solution using a peristaltic pump. It was operated under “once through” mode, i.e. the substrates entered the pores of the membrane, converted to the products, and the product stream exited the device. Immobilized enzymes catalyzed the reaction between glucose and O2 to form gluconic acid and H2O2, and H2O2 was then analyzed to quantify performance of the immobilized enzyme. Glucose solution of 15 mM concentration, saturated with O2, was used as substrate solution for the activity measurement experiments of the immobilized GOx enzyme. For comparison between convective and diffusive flows, a GOx-immobilized biomimetic membrane was subjected to (i) diffusive flow by submerging the membrane in oxygen saturated 3 ml of 15 mM glucose solution (batch reactor), and (ii) convective flow by passing oxygen saturated 15 mM glucose solution (continuous reactor). Further details of activity and stability analysis for the PLB functionalized GOx-immobilized membranes are reported elsewhere
24.
Here, we focus on
exploring the reusability of the functionalized membrane and the phospholipid.
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ECH solution
+ + + ++
PEI solution
+ + + ++
GOx solution
SUV solution
NH
NH2
NH
H2C CH CH2
H2 C
OH NH + 2 PEI
Bare nylon membrane
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CH
CH2
OH
+ NH2
NH
Nylon-ECH-PEI-PLB (PLB-functionalized) membrane
Nylon-ECH-PEI membrane
H2 C
DMPC
CH
CH2
OH
+ NH2
DMPC
GOx
Nylon-ECH-PEI-PLB-GOx (PLB functionalized GOX immobilized) membrane
Dilute HCl at pH 3.5
+ + + ++ + + + ++
+
Nylon-ECH-PEI membrane
1 M NaCl, pH 11
SUVs in solution
+
Nylon-ECH-PEI-PLB (PLBfunctionalized) membrane
Enzyme in solution
Figure 2. Schematic representation of formation of reversibly attached phospholipid bilayer (PLB) functionalized nylon membrane pore containing electrostatically immobilized GOx followed by sequential detachment of GOx and PLB. ECH – epichlorohydrin, PEI – polyethyleneimine, PLB – phospholipid bilayer, SUVs – small unilamellar vesicles, GOx – glucose oxidase enzyme. 2.2.6. Reusability of the functionalized membrane and phospholipid: Reusability of the functionalized membrane and phospholipid was explored following a sequence of “attachmentdetachment-reattachment”. The sequential attachment-detachment steps due to electrostatic interactions between the components are depicted in Figure 2. To elute GOx, an eluent solution of dilute HCl with pH 3.5 was permeated through the membrane. PLB within the membrane remained intact as it was electrostatically attached to the PEI cushion under the above mentioned condition. Therefore, PLB functionalized membrane could be used repeatedly for enzyme immobilization. This approach of detachment of the electrostatically immobilized enzyme is useful to remove the deactivated enzyme and replenish the same membrane matrix with fresh enzyme. Alternatively, it can be implemented to immobilize different enzymes for different applications without changing the membrane matrix. After enzyme detachment, the PLB was detached by permeating an eluent solution (1 M NaCl, pH 11). This resulted in the detachment of the PLB as demonstrated in Figure 2. 8 ACS Paragon Plus Environment
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2.3. Analytical procedures: For all spectrophotometric measurements of various liquid samples, Ultraviolet-Visible (UV-Vis) Spectrophotometer (Varian, Cary 60) was used. 2.3.1. Analysis of phospholipid: To quantify phospholipid, Stewart Assay
32
was used.
The assay is based on the formation of a complex between phospholipids and ammonium ferrothiocyanate reagent. 1 mL of thiocyanate reagent was added to 1 mL of phospholipidchloroform mixture. After vortexing for 1 min, lower red layer was removed and absorbance was taken at 488 nm. Concentration of phospholipid was determined with the help of a standard calibration curve. The linear range for Stewart Assay was 10-100 μg/mL in this study. 2.3.2. Analysis of enzyme: For the quantification of GOx, Bradford Protein Assay was used
33.
The procedure of Bradford Protein Assay is based on the formation of a protein-dye
complex between protein in solution and a dye, Coomassie Brilliant Blue G-250. The absorption maximum of the dye shifts from 465 to 595 nm after the formation of protein-dye complex. 0.1 mL of enzyme sample was mixed with 3 mL of Bradford reagent, vortexed and incubated at room temperature for 10 min. Then, absorbance was taken at 595 nm. The concentration of enzyme was determined with the help of a standard calibration curve, which was prepared from standard BSA solutions. The linear range of the Bradford Protein Assay was 0.1-1.4 mg/mL in this study. The amount of immobilized enzymes was determined by analyzing the concentration of initial enzyme solution, concentration of enzyme solution after immobilization and concentration of enzyme in washing solution. 2.3.3. Analysis of Hydrogen peroxide (H2O2): A colorimetric method described by Eisenberg 34 was used to analyze the concentration of H2O2 in solution. The assay is based on the spectrophotometric measurement of the color intensity of H2O2 solution treated with titanium oxysulfate reagent to form pertitanic acid (TiO2.H2O2). To analyze H2O2 sample, 100 µl titanium oxysulfate was added to 900 µl sample. Mixture was vortexed and absorbance was taken at 407 nm. The concentration was determined with the help of a calibration curve, which was prepared from standard H2O2. The linear range of H2O2 analysis by this colorimetric method was 0.03-3 mM in this study.
3. Results and discussion:
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3.1. Characterization of SUVs: Liposomes are spherical structures of phospholipids with size range between 10 nm and 1000 nm. In this study, they were formed by mixing phospholipids in chloroform followed by forming a clear lipid film by subsequent removal of chloroform. This thin lipid film, when hydrated, became swollen. Then, under the influence of agitating force, the hydrated and swollen lipid film spontaneously converted into vesicles of various sizes. These vesicles were then disrupted using sonication to form SUVs 28. SUV solution, which was used for development of functionalized biomimetic membrane, was characterized by Dynamic Light Scattering (DLS). Average hydrated diameter of the SUVs was observed to be around 90 nm as demonstrated in Figure 3. Point to be noted that SUV diameter of
