Article Cite This: ACS Appl. Bio Mater. 2019, 2, 2453−2463
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High Permeability of Polyunsaturated Lipid Bilayers As Applied to Attoliter Enzyme Reactors Naoyuki Nagatomo and Makoto Yoshimoto* Department of Applied Chemistry, Yamaguchi University, Tokiwadai 2-16-1, Ube 755-8611, Japan
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ABSTRACT: Phospholipid vesicles encapsulated with enzymes have potential applications for artificial organelles. A critical problem associated with the compartmentalized enzymes is their low reactivity because of the permeability resistance of lipid bilayers to substrates. In the present work, the polyunsaturated bilayers of 1,2-dilinolenoyl-snglycero-3-phosphocholine (18:3-PC) were elucidated to be highly permeable to 5(6)-carboxyfluorescein at high temperatures up to 60 °C and applied to fabricate vesicle-based reactive enzyme reactors. D-Amino acid oxidase (DAO) from porcine kidney was encapsulated in 18:3-PC vesicles with each aqueous volume of 3.4 × 10−21 m3 (=3.4 aL). The DAO-containing vesicles were highly reactive at 40 °C toward D-alanine being added to bulk solution at pH 9.0 and stably catalyzed following two types of reactions. One is the DAO-catalyzed continuous production of H2O2 in the vesicles for 30 min being detected by the free peroxidase-catalyzed oxidation of o-dianisidine in bulk solution. The other is the cascade reaction in the vesicles coencapsulating DAO and catalase being followed for 5 h on the basis of the concentration of unreacted D-alanine. In the latter reaction, the intermediate product H2O2 was decomposed by catalase producing oxygen allowing its cyclical use for the DAO-catalyzed oxidation. Furthermore, thanks to the highly temperature-dependent permeability of 18:3-PC bilayers, on/off-like switching in the activity could be induced with respect to the vesicle-confined enzyme by shifting the reaction temperature between 20 and 40 °C. The above reactive vesicles can offer the opportunity of the H2O2-based reliable detection of D-amino acids and the continuous optical resolution of racemic mixtures of amino acids. KEYWORDS: liposomes, polyunsaturated lipids, bilayer permeability, D-amino acid oxidase, catalase, peroxidase
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INTRODUCTION Assemblies of amphiphilic molecules such as synthetic block copolymers and phospholipids with long acyl chains have received more and more attention because of the similarity to living organelles in their scale, modifiability of membranes, and the ability to stably encapsulate enzymes.1−4 Polymersomes and phospholipid vesicles or liposomes are well-characterized assemblies which offer the opportunity to spatially confine catalytically different enzymes for a cascade reaction5−10 or to isolate single enzyme molecules for their thermal stabilization.11 These systems are also applicable to fabricate multicompartmentalized soft materials like vesosomes12 for achieving spatially controlled encapsulation of different compounds and chemical reactions.13 Among molecular assemblies, phospholipid vesicles with high biocompatibility are suitable for developing functional drug carriers and materials mimicking biological membranes. Lipid vesicles prepared with the well-known extrusion technique14,15 possess typical diameters of several hundred nanometers. If one assumes the diameter of a vesicle of 200 nm, it gives a very large surface-to-volume ratio of 3 × 107 m−1 and an aqueous volume of ≈4 × 10−21 m3 (=4 aL (attoliter)) capable of solubilizing enzymes. For such colloidal enzyme reactors, the © 2019 American Chemical Society
transfer process of substrates from bulk solution to the water pool of reactors becomes of great importance because the mass transfer process often controls the overall rate of reactions.13,16 It is, therefore, desired for practical applications that compartmentalized enzymes with high reactivity are prepared through increasing the permeability of lipid bilayers without employing molecular machineries such as channel-forming membrane proteins. Physicochemical properties and functions of bilayer membranes are related to the degree of unsaturation of lipids17−21 or bilayer-forming polyunsaturated fatty acids (PUFAs).22,23 Fluidity and elasticity of lipid bilayers are affected by the degree of unsaturation of lipids.24,25 It was also reported that the mixture of phospholipids with oleic, linoleic, and linolenic acids, which were identical in the number of carbon atoms of 18 and different in the number of double bonds in each acyl chain, could contribute to form adequately fluid bilayers of soya lechitin.26 These previous observations suggest that polyunsaturated lipids can be the candidates for Received: February 25, 2019 Accepted: May 17, 2019 Published: May 17, 2019 2453
DOI: 10.1021/acsabm.9b00165 ACS Appl. Bio Mater. 2019, 2, 2453−2463
Article
ACS Applied Bio Materials the preparation of highly permeable bilayers. In this context, the effects of chain length and degree of unsaturation of lipids need to be elucidated on bilayer permeability at different temperatures. D-Amino acid oxidase (DAO) is the flavoenzyme that catalyzes the oxidation of various D-amino acids producing H2O2 and corresponding imino acids being hydrolyzed giving α-keto acids and the ammonium ion.27,28 Free DAO is functionally fragile as reported previously.16,29,30 Immobilized DAO is useful for biochemical and bioanalytical applications, for example, for detecting clinically important D-serine.31,32 The DAO molecules were encapsulated in phosphatidylcholine-based vesicles to examine the permeability of various amino acids through mixed lipid bilayers.33 We reported that the oxidation of D-amino acids catalyzed by lipid vesiclesencapsulated DAO was decelerated compared to free-enzymecatalyzed reactions because of the permeation resistance of bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) toward the substrates such as D-serine and 16 D-alanine. Thus, the vesicles can be utilized to apparently alter the substrate selectivity of DAO on the basis of the intrinsic bilayer permeability of each substrate.16 Furthermore, the storage stability of DAO in vesicles at 4 °C was reported to be higher than that in POPC/detergent mixed micelles.16 The DAO-containing vesicles are potentially applicable to the quantification of D-amino acids on the basis of H2O2 produced in the oxidation reactions and to the compartmentalization of enzymes for catalyzing cascade reactions. For these types of applications, the reactivity of liposomal DAO needs to be controllable. In particular, the vesicles with high bilayer permeability are of significance for achieving both sufficient reactivity and functional stability of the compartmentalized enzyme. In this work, polyunsaturated phospholipid vesicles were applied to fabricate highly reactive compartmentalized DAO. The chemical structure of lipids used is shown in Figure 1. The
Figure 2. Schematic overview of the present work. (A) Vesicles encapsulated with 5(6)-carboxyfluorescein (trianion) (VET-CF) (VET: vesicles prepared by the extrusion technique,14,36 see the Experimental Section). VET-CF was prepared and used for the selection of highly permeable lipid bilayers. (B) Two types of the vesicles-based enzyme reactors with attoliter-scale aqueous volumes. One of the two reactors is (i) the vesicles encapsulated with D-amino acid oxidase (VET-DAO), and the other is (ii) the vesicles coencapsulated with D-amino acid oxidase and catalase (VET-DAO/ CAT). DAO from porcine kidney is a dimer, and each subunit contains a noncovalently bound flavin adenine dinucleotide (FAD) molecule.27 In both reactors, the D-alanine (D-Ala) molecules, which are initially present in the bulk solution at pH 9.0, pass through lipid bilayers followed by the DAO-catalyzed oxidation of D-alanine producing pyruvate, ammonium ion, and H2O2. In the VET-DAOcatalyzed reactions, H2O2 can be accumulated in the vesicle suspensions, while in the VET-DAO/CAT-catalyzed reactions, the catalase (CAT) molecules offer the opportunity to decompose H2O2 produced by the DAO-catalyzed reaction yielding molecular oxygen. For the chemical structure of phospholipids used (18:1-, 18:2-, and 18:3-PC), see Figure 1.
hydrophilic compound, 5(6)-carboxyfluorescein (CF) (Figure 2A). The release of CF from vesicles was kinetically analyzed to quantitatively evaluate the bilayers on the basis of the permeability coefficient of CF, PCF.34,35 Then, 1,2-dilinolenoylsn-glycero-3-phosphocholine (18:3-PC) bilayers, which were revealed to be highly permeable to CF, were applied to the compartmentalization of DAO for fabricating the vesiclesbased enzyme reactors (see Figure 2Bi). Not only the reactivity of compartmentalized DAO in the vesicles but also the difference in the heat stability between the compartmentalized and free enzyme was examined in detail. Furthermore, the polyunsaturated vesicles were applied to encapsulate the DAO and catalase molecules to fabricate an attoliter reactor for a catalytic cascade reaction (see Figure 2Bii).
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Figure 1. Chemical structure of polyunsaturated phospholipids used. “18:1-PC”, for example, means phosphatidylcholine (PC) possessing diacyl chains with the number of carbon atoms of 18 and the number of double bonds of 1 in each chain.
EXPERIMENTAL SECTION
Materials. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (18:1-PC) (commercial name: MC-8181 (DOPC), lot 1504811) was obtained from NOF (Tokyo, Japan). 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (18:2-PC) (lot 850385-01-192A) and 1,2-dilinolenoyl-sn-glycero3-phosphocholine (18:3-PC) (lot 850395C-25MG-G-123 and lot 850395-200MG-I-123) were obtained from Avanti Polar Lipids. 5(6)Carboxyfluorescein (CF) (lot BCBQ7825 V) and D-amino acid oxidase from porcine kidney (DAO) (catalog A5222, lot SLBT8735, lot SLBT2257, and lot SLBX2422) were obtained from SigmaAldrich. Bovine liver catalase (CAT) (lot WDQ3945), horse radish peroxidase (HRP) (lot LKE0277), and acetone (>99.8%, lot
lipids are phosphatidylcholines each of which possesses two identical acyl chains with the number of carbon atoms of 18. The lipids with the number of double bonds in each acyl chain of 1, 2, and 3 are abbreviated in this work as 18:1-PC, 18:2-PC, and 18:3-PC, respectively. An overview of the work presented is schematically shown in Figure 2. The effect of the degree of unsaturation of phospholipids on their bilayer permeability was first examined using the vesicles encapsulated with a model 2454
DOI: 10.1021/acsabm.9b00165 ACS Appl. Bio Mater. 2019, 2, 2453−2463
Article
ACS Applied Bio Materials ECP3897) were obtained from Wako Pure Chemical Industries. 3Amino-1,2,4-triazole (3-AT) (lot WMHBA-JM) was obtained from TCI. D-Alanine (lot SLBC1877 V), o-dianisidine dihydrochloride (lot SLBT8735), fluorescamine (lot 015M4047 V), and sepharose 4B gel beads (lot MKBX9438 V) were obtained from Sigma-Aldrich. All lipids, enzymes, and chemicals were used as received. Water used was purified and sterilized with a water purification system, Elix Essential UV3, from Merck. Preparation of 5(6)-Carboxyfluorescein-Containing Vesicles (VET-CF). In this work, all vesicles were prepared by hydrating a thin lipid film followed by freeze−thaw treatments and extrusion for sizing. These vesicles are abbreviated as VET (vesicles prepared by the extrusion technique).14,36 For the preparation of VET-CF, each lipid (25−50 mg) was dissolved in a 100 mL round-bottom flask with 4 mL of chloroform, and the solvent was removed by using a rotary evaporator. The lipid was dissolved with 4 mL of diethyl ether, and the solvent was removed. This process was performed two times. A thin lipid film formed and was kept under high vacuum (
