Langmuir 1995,11, 4013-4018
4013
Covalent Immobilization of Polymeric Bilayer Membranes to Porous Supports Masa-aki Wakita* and Masanori Hashimoto Central Laboratories, Kurita Water Industries, Ltd., 7-1 Wakamiya, Morinosato, Atsugi-city, Kanagawa 243-01, Japan Received March 13, 1995. In Final Form: June 12, 1995@ Chitosan was reacted with 1-bromooctadecaneto yield N-octadecylchitosanconsisting of 70 mol % of 2-(octadecylamino)-2-deoxy-~-glucopyranose, 17 mol % of 2-amino-2-deoxy-D-glucopyranose (GlcN), and 13 mol % of 2-acetamido-2-deoxy-~-glucopyranose. N-Octadecylchitosan shows a gel to liquid-crystalline phase transition in a differential scanning calorimetry (DSC, T,= 46 "C) and forms bilayer membranes with 50 thickness (electron microscopy) in acidic water by sonication. Polymeric bilayer membranes of N-octadecylchitosan were covalently immobilized to carboxylated porous supports by amidation between primary amino groups of GlcN moieties and carboxyl groups of the supports. Electron microscopy and DSC of the supports suggested the retention of membrane structure upon the immobilization. The resulting porous supports bearing polymeric bilayer membranes are stable to 0.5 N NaOH or 0.1N HC1. The amount of the released organic carbon from them during a washing with water for 24 h was less than 0.04% of the organic carbon of the immobilized N-octadecylchitosan.
Introduction Lipid bilayer membranes have been receiving considerable attention as models for various biochemical investigations such as permeability contro1,l chemical sensing,2 and separation of protein^.^ For these studies, a n immobilized form of membrane with enhanced stability is favorable rather than a vesicular dispersion. Substantial efforts have been done for the stabilization and the immobilization of lipid membranes. For example, polymerization of lipid molecules in bilayer vesicles4and vesicle formation from polymeric amphiphiles5have been reported to enhance the stability of membrane structure. Lipid bilayer membranes were immobilized as composite films with polymers: and ionic complexeswith polyele~trolytes.~ Phospholipid liposomes were entrapped in pores of chromatographic gel beads.8 Vesicles composed of amphiphilic amino acid derivatives were immobilized to chromatographic gel beads by covalent bonds between part of lipid molecules in vesicles and the gel beads.g By these methods lipid bilayer membranes were immobilized with retention of their characteristics such as a gel to liquidcrystalline phase transition. However, elution of lipid molecules is inevitable under uses in a n aqueous phase, because they, at least some of them, are not covalently immobilized to support materials. Chemical stability of immobilized lipid membranes can be a crucial issue for practical uses. For example, pyrogenic lipopolysaccharide (LPS), a contamination in Abstract published in Advance A C S Abstracts, September 15, 1995. (1)(a) Okahata, Y. Ace. Chem. Res. 1986,19,57. (b) Okahata, Y.; and references Ariga, K.; Seki, T. J.Am. Chem. SOC.1988,110,2495, therein. (2)(a)Okahata, Y.; Shimizu,0.Langmuir 1987,3,1171.(b)Nikoleis, D. P.; Krull, U. J. Electroanalysis 1993,5,539, and references therein. (3)Lundahl, P.; Yang, Q. J. Chromatogr. 1991,544,283. (4) For a review, see: (a)Regen, S. L. InLiposomes: From Biophysics to Therapeutics: Ostro, M. J., Ed.; Marcel Dekker: New York, 1987; p 73. (b) OBrien, D. F.; Ramaswami, V. In Encyclopedia of Polymer Science and Engineering, 2nd ed.; Mark, H. F., Bikales, N. M., Overberger, C. G., Menges, G., Kroschwitz, J. I., Eds.; John Wiley & Sons: New York, 1989;Vol. 17;p 108. ( 5 ) Kunitake, T.; Nagai, M.; Yanagi, H.; Takarabe, K.; Nakashima, N. J. Macromol. Sci.-Chem. 1984,A21 (8&9),1237. (6)Shimomura, M.; Kunitake, T. Polym. J. 1984,16,187. (7)Kunitake, T.; Tsuge, A.; Nakashima, N. Chem. Lett. 1984,1783. (8)Yang, Q.;Lundahl, P. Anal. Biochem. 1994,218,210. (9)Neumann, R.; Ringsdorf,H.; Patton, E. V.; O'Brien,D. F. Biochim. Biophys. Acta 1987,898,338. @
Scheme 1
Activated Porous SUDDOflS
Polymeric Bilayers
Condensation
with
Functional Groups
protein solutions, has to be removed for a pharmaceutical use of proteins. We have reported that rigid bilayer membranes adsorb LPS selectively from protein solutions and conventional octadecyl ligands adsorb both LPS and proteins.1° To use porous supports having immobilized lipid bilayer membranes for a chromatographic removal of LPS, elution of lipids should be minimal to prevent contamination of proteins by lipids. The stability under a sterilization process, which is usually done in protein purification by (treatingchromatographic gel beads with NaOH solution,ll is also essential. Various activated lipids have been prepared and reacted with support materials to yield covalently immobilized lipid monolayers12 and not bilayers. Thus, preparation of covalently immobilized lipid bilayer membranes with substantial chemical stability is interesting as well as practically important. In this paper we report the covalent immobilization of lipid bilayer membranes to porous supports. Scheme 1 shows our concept that lipid bilayer membranes are prepared from polymeric amphiphiles in aqueous phase and covalently immobilized to a porous support at their hydrophilic moieties. Advantages of this methodology are (i) a use of polymeric amphiphiles completes bilayer formation prior to a n immobilization process and (ii) (10)Wakita, M.-a.; Hashimoto, M. Langmuir 1996,11, 607. (11)Process Chromatography A Practical Guide; Sofer, G. K., Nystrom, L.-E., Eds.; Academic Press: London, 1989;p 95. (12)(a)Okahata, Y.; Ariga, K.; Shimizu, 0.Langmuir 1986,2,538. (b) Pidgeon, C.;Venkataram, U. V. Anal. Biochem. 1989,176,36.(c) Kallury, K.M. R.; Lee, W. E.; Thompson, M. Anal. Chem. 1992,64, 1062.
0743-7463/95/2411-4013$09.00/00 1995 American Chemical Society
4014 Langmuir, Vol. 11, No. 10, 1995
Chart 1
GlcNC18 I
NH(CH2)17CH3
GlcN
GlcNAc NHCOCH,
because all lipids molecules are covalently immobilized t o the s u p p o r t t h r o u g h polymer main chains, the immobilized bilayer membranes are stable and suitable for various practical applications. Furthermore, conformation and mobility of the immobilized polymeric amphiphiles, which influence properties of their membranes, might be controlled b y factors s u c h as a molecular weight of the amphiphile and an a m o u n t of condensation reagents for the immobilization. F o r a first example, by this methodology, a novel polymeric amphiphile was designed from chitosan. Chit o s a n is a polymer obtained by deacetylation of chitin, and consists of p-1-4-linked 2-amino-2-deoxy-D-glucopyr a n o s e (GlcN, Chart 1 ) and 2-acetamido-2-deoxy-~-glucopyranose (GlcNAc). Partial N-octadecylation of chitosan produces a polymeric amphiphile which contains 24octadecylamino)-2-deoxy-~-glucopyranose (GlcNC 181, residual GlcN having a reactive p r i m a r y a m i n o group for a covalent immobilization, and GlcNAc. Bilayer membranes were prepared from N-octadecylchitosan and immobilized t o carboxylated porous s u p p o r t s using carbodiimides. The resulting porous s u p p o r t s bearing polymeric bilayer membranes were characterized w i t h IR spectroscopy, electron microscopy, and DSC.
Experimental Section Materials and General Methods. Chitosan was purchased from Dai-ichi Kogyo Seiyaku Co., Ltd. (Kyoto, Japan). Degree of deacetylation was determined as 87 mol % by colloidal titration.13 Intrinsic viscosity was 1.42 d u g (0.2 M CH3COOW 0.1 M CH&OONa, 30 "C) which corresponded to 2.67 x lo4 of molecular weight relative to poly(ethy1ene glycol).14 Kurimover 11, a product of Kurita Water Industries, Ltd. (Tokyo, Japan), is a cross-linked porous chitosan gel particle having a particle size of 45-420pm and an average pore diameter of 2 pm. 1-Bromooctadecane, succinic anhydride, acetic anhydride (Kishida Chemical Co., Ltd., Osaka, Japan), l-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (WSC, Dojindo Laboratories, Kumamoto, Japan), N-hydroxysuccinimide (HOSu), and D-(+)glucosamine hydrochloride (Tokyo Kasei Kogyo Co., Ltd., Tokyo) were used as obtained. Avolume of gel particles was determined after standing for 24 h in a measuring cylinder with water. IR and 'H NMR spectra were recorded on a IR-810 (Jasco Co., Ltd., Tokyo) and a JNM-GX270 (JEOL, Ltd., Tokyo), respectively. Elemental analysis, total organic carbon content analysis, and DSC were carried out using a MT-3 (YanagimotoMFG. Co., Ltd., Tokyo), a TOC-5000A(Shimadzu MFG., Kyoto), and a DSC220C (Seiko Instruments Inc., Tokyo), respectively. Sonication was performed with a probe type sonicator UD-201 (Tomy Seiko, (13)Terayama, H. J.Polym. Sci. 1952, 8, 243. (14)Relationship between intrinsic viscosity and molecular weight of chitosan was determined by GPC. Unpublished results.
Wakita and Hashimoto Tokyo). A dynamic light scattering measurement was carried out using a DLS-700 (Otsuka Electronics Co., Ltd., Osaka) at 20 "C with an angle of 90". Transmission electron micrographs (TEM) and scanning electron micrographs (SEM)were taken a t JEOL DATUM (Tokyo) using a JEM-1200EX and a JSM-6400F (JEOL),respectively. An ultrathin section ofN-octadecylchitosan dispersion was prepared according to Luft's method15using Os01 for staining, agar and acetone for dewatering, epoxy resin (Epon 812) for embedding, and uranyl acetate and lead citrate for final staining. For N-octadecylchitosan-immobilizedsupports and carboxylated ones, ethanol and propylene oxide were used for dewatering. N-Octadecylchitosan. Chitosan (5.00 g a s product including 9.1% of moisture and 0.4% of inorganic matter) was dissolved to aqueous acetic acid solution and precipitated by neutralization with NaOH. After being washed by water and dimethylacetamide successively, chitosan was reacted with 1-bromooctadecane (23.51 g, 70.6 mmol) in the presence of Na2C03 (7.48 g, 70.6 mmol) a t 70-75 "C for 144 h in dimethylacetamide. The reaction mixture was poured into water and the resulting precipitates were washed with water, hot ethyl acetate, water, and acetone successively. Subsequent vacuum drying yielded 9.55 g of product. lH NMR (dispersion in D20/DMSO-d6, l/9 (v/v), including DC1): 6 0.8 (GlcNC18, CH3), 0.9-1.9 (GlcNC18, (CH2)16), 2.05 (GlcNAc, N(C=PO)CH~), 2.7-3.0 (GlcNC18, N-CHz), 3.0-4.2 (GlcNC18, GlcN, GlcNAc, ring C2-C6 proton, OH), 4.4-4.6 (GlcNAc, ring C1 proton), 4.7-5.2 (GlcNC18, GlcN, ring C1 proton). Degree of N-octadecylation determined from the ratio of N-CHZ (GlcNC18) to ring C1 proton (GlcNC18, GlcN, GlcNAc) is 70 mol %. IR (mrpellet): YCH Of CH2 2925,2850 cm-', ~ C O Hf CH2 1470, 720 cm-'. Anal. Calcd for 70 mol % GlcNC18,17 mol % of GlcN, and 13 mol % of GlcNAc: C, 66.0; H, 10.7; N, 4.1. Found: C, 65.7; H, 10.5; N, 3.9. Preparation of Carboxylated Porous Supports. Kurimover I1 (37.0 mL, including 8.4 mmol of NH2) was washed by water and equilibrated with 10% aqueous acetic acid. The resulting gel particles were washed with 10% aqueous acetic acidmethanol (l/4 (v/v)) and reacted with succinic anhydride (2.54 g, 25.3 mmol) in 10% aqueous acetic acidmethanol (1/4 (v/v))at 60 "C for 5 h. Then acetic anhydride (1.6 mL, 16.9 mmol) was added to the mixture and reaction was continued at 40 "C overnight. The resulting gels were washed by 10% aqueous acetic acidmethanol (l/4 (v/v)), methanol, acetone, and water successively. Preparation of Bilayer Dispersion of N-Octadecylchitosan. N-Octadecylchitosan (20 mg) was dispersed to 20 mL of water including lOOpmol of HCl by vortex mixing and subsequent sonication at ca. 70 "C for 10 min. Aqueous NaOH was added to adjust pH to 6.4 and the obtained dispersion was filtered through a Millex-SV filter unit having 5.0-pm pore diameter (Millipore Co.) for a dynamic light scattering and TEM observations. For an immobilization to carboxylated supports, 200 mg ofN-octadecylchitosan was dispersed to 50 mL ofwater including 700 pmol of HC1 similarly. Immobilization ofN-Octadecylchitosanto Carboxylated Porous Supports. The carboxylated porous support (12.2 mL) was suspended in water and the pH of the suspension was adjusted to 7.0 by NaOH and HC1 aqueous solution. The support gel particles were recovered by filtration and added to 50 mL of N-octadecylchitosan bilayer dispersion (pH 6.9, 4 mg/mL). A 340-pL aliquot of WSC/HOSu aqueous solution (20/25 mg per mL) was added and pH was adjusted to 6.8. After being shaken overnight a t 20 "C, the gel particles were recovered and washed withO.l N HCl and water successively. The resultinggelparticles were immersed in 0.5 N NaOH overnight and then washed with water, 0.1 N HC1, and water successively. This alkali treatment was done to hydrolyze possible ester linkages between carboxyl groups ofthe support and hydroxyl groups ofN-octadecylchitosan. Two identical batches of these gels were collected and added to a 35-mL aqueous solution (pH 6.9) of D-(+)-glucosamine hydrochloride (3.83 g, 17.8 mmol) including WSC (2.27 g, 11.9 mmol). HOSu (1.36 g, 11.9 mmol) was added to the mixture and reacted overnight a t 20 "C. After a washing with water, the gel particles were immersed in 0.5 N NaOH overnight and washed thoroughly with water, 0.1 N HC1, and water successively. (15) Luft, J. H. J.Biophys. Biochem. Cytol. 1961,9,409.
Covalent Immobilization of Bilayer Membranes
Langmuir, Vol. 11, No. 10, 1995 4015
Figure 1. Transmission electron micrograph of bilayer dispersion ofN-octadecylchitosan (ultrathin section), x 200000 as provided. Chemical Stability. N-Octadecylchitosan-immobilized gel particles were immersed in 0.5 N NaOH or 0.1 N HC1 at ambient temperature overnight and then washed with water and subjected to IR spectroscopicanalysis. N-Octadecylchitosan-immobilized gel particles (20 mL) were packed to a column (Pharmacia LKl3 Biotechnology, XK16/20, 16 mm i.d. x 100 mm length). The packed column was filled with 0.5 N NaOH and allowed to stand overnight a t 20 "C. After a washing with 200 mL of water, 50 mL of water was circulated in the column for 24 h a t a flow rate of 1BVh. The water was recovered and subjected to a n analysis of total organic carbon content.
Results and Discussion Synthesis of N-Octadecylchitosan. Chitosan composed of 87 mol % of GlcN and 13 mol % of GlcNAc was simply N-alkylated by l-bromooctadecane to yield Noctadecylchitosan consisting of 70 mol % of GlcNC18, 17 mol % of GlcN, and 13 mol % of GlcNAc. Bilayer Formation from N-Octadecylchitosan. A dispersion was prepared by suspending N-octadecylchitosan in water including hydrochloric acid (vortex mixing) and successive sonication at ca. 70 "C. A dynamic light scattering measurement indicated the existence of particles haviqg diameters of 110 f 10, 670 f 80, and 3090 f 670 A. TEM of a ultrathin section of the centrifbged dispersion revealed the membraneous structure with ca. 50 A thickness and various lengths (Figure 1).16 Considering that molecular weight of chitosan used in this study is 2.67 x lo4relative to poly(ethy1ene glycol), (16)In Figure l,OsO* was used for staining to compare with TEM images of the thin section of the porous supports bearing N-octadecylchitosan membranes. We observed typical vesicle structures with distinct walls of similar thickness in a N-octadecylchitosan dispersion negatively stained with uranyl acetate.
Scheme 2 Succinic Anhydride
Porous Supports (Chitosan~ ~ 1CH3COOHaq/CH30Hb )
Carboxylated
KOctadecvlchitosan Bilayer Dispersion supports
b
WSC/HOSu D-(+)-Glucosamine WSC/HOSu
N-Octadecylchitosan Bilayer membrane immobilized supports
these results clearly show that N-octadecylchitosan forms molecular assemblies in a n aqueous phase. The thickness of the membrane layers observed in TEM is typical of lipid bilayer membrane. In DSC of N-octadecylchitosan, a broad but definite endothermic peak was observed (T, = 46 "C) indicating a gel to liquid-crystalline phase transition.
Covalent Immobilization of N-Octadecylchitosan to Carboxylated Porous Supports. Kurimover 11, a cross-linked porous chitosan gel particle, was used as a support because of its chemical stability and aggregateaccessible macropores. The synthetic routes are outlined in Scheme 2. To introduce carboxyl groups, Kurimover I1 was treated with excess succinic anhydride in aqueous acetic aciamethanol mixed solvent. Acetic anhydride was subsequently added to the reaction mixture to reduce residual primary amino groups on the surface. An IR spectrum of the resulting gel particles (Figure 2) showed a new absorption band at 1720 cm-l (VC=O of COOH) and
4016 Langmuir, Vol. 11, No. 10, 1995
Wakita and Hashimoto
4000
3000
2000
1500
1000
500
Wavenumber (cm-1) 1655 cm-I
4000
3000
2000
1560 cm-1
1500
Wavenumber (cm-1)
1000
500
Figure 3. IR spectra: (a) difference spectrum, N-octadecylchitosan-immobilized supports minus carboxylated supports; (b) N-octadecylchitosan.
,
Figure 2. IR spectra of (a) porous supports, (b) carboxylated porous supports, and (c) N-octadecylchitosan-immobilizedsupports.
an increase of intensity at 1655,1560 cm-l (YC=O of NHCO) indicating selective N-su~ciny1ation.l~ The obtained carboxylated supports were reacted with membraneousN-octadecylchitosanin the presence of WSC and HOSu. The resulting gel particles were washed using 0.1 N HC1 to elute unimmobilized N-octadecylchitosan and adsorbed one by ionic interaction which might exist. Residual carboxyl groups on the surface were amidated with D-(+)-glucosamine. The differencein IR spectra upon the immobilization was a small but distinct increase of YCH a t 2925 cm-l and a disappearance of vc-0 of COOH at 1720 cm-l (Figure 2). The increase of YCH can be assigned to N-octadecylchitosan methylenes, and the disappearance of YC=O is due to amides formations from carboxyl groups of the supports and primary amino groups of N-octadecylchitosan and D-(+)-glucosamine. The difference spectrum, N-octadecylchitosan-immobilized supports minus carboxylated ones, clearly demonstrates VCH of N-octadecylchitosan methylenes at 2925, 2850 cm-l, and YC=O of NHCO at 1655,1560 cm-l (Figure 3). These are direct evidence for the covalent immobilization of N-octadecylchitosanto the carboxylated supports by amide bonds. In a titration curve of the N-octadecylchitosanimmobilized supports using 0.1 N HC1, consumption of HC1 was observed from pH 9.0 indicating a dissociation of secondary amino groups. In the case of Kurimover I1 having only primary amino groups, consumption of HC1 was observed from pH 7.4. The amount of immobilized N-octadecylchitosan was estimated as a 4 mg/mL gel assuming that the amount of HC1 consumed a t pH 9.07.4 was attributable to GlcNC18. Structure of the Immobilized N-Octadecylchitosan. SEMS of the carboxylated supports and the Noctadecylchitosan-immobilizedones were shown in Figure 4. The N-octadecylchitosan-immobilizedsupports showed porous structure, and their surface was rougher than that of the carboxylated one indicating the existence of Noctadecylchitosan. To see detailed structure of the im(17) Hirano, S.;Moriyasu, T.Curbohydr.Res. 1981,92,323.
Figure 4. Scanning electron micrographs of (a)N-octadecylchitosan-immobilized supports and (b) carboxylated supports, x20000 as provided.
mobilizedN-octadecylchitosan,ultrathin sections of those supports were observed by TEM (Figure 5). Interestingly in the case of the N-octadecylchitosan-immobilized support, threadlike materials were observed around the fibrous support. Os04 was used as a staining reagent to prepare ultrathin sectionsin this work. Ammonium lipids
Covalent Immobilization of Bilayer Membranes .
.--
a
Langmuir, Vol. 11, No. 10, 1995 4017
--
Figure 5. Transmission electron micrographs of (a) N-octadecylchitosan-immobilized supports and (b) carboxylated supports (ultrathin section), x 60000 as provided.
can be stained with Os0418 as well as unsaturated materials. The threadlike materials are cationic Noctadecylchitosan aggregates which interact with anionic 0 ~ 0 4 The . sizes of the threads, which are roughly in order of tens of angstroms thickness and hundreds to one thousand angstrom length, are similar to those of Noctadecylchitosan bilayer membranes shown in Figure 1. DSC thermograms of dried N-octadecylchitosan-immo(18)Riemersma, J. C.Biochim. Biophys. Acta 1968,152,718.
bilized supports and N-octadecylchitosan are shown in Figure 6. Phase transition was not observed with Noctadecylchitosan-immobilizedsupports. However, when the immobilized supports were heated at 60 "C in acidic water prior to drying, a slight endothermic phenomenon centered at ca. 37 "C was observed. This heat effect was reproducible. Peak area ratio of N-octadecylchitosanimmobilized supports (b in Figure 6) to N-octadecylchitosan ( c ) is 7% and similar to the w t % of N-octadecylchitosan in the immobilized support (6%). Although a
Wakita and Hashimoto
4018 Langmuir, Vol. 11, No. 10, 1995
v
+ 46
0
20
40
'C
60
80
100
Temperature ('C)
Figure 6. DSC thermograms (heating rate, 1.0 "C/min): (a) N-octadecylchitosan-immobilized supports; (b) N-octadecylchitosan-immobilizedsupports after being heated at 60 "C for 1h in water (pH 2.7, HCI);(c)N-octadecylchitosan. For (a)and (b),porous supports without chemical modification were used
as reference. DSC curve is not clear, the heating in acidic water seems to develop bilayer structures of the immobilized Noctadecylchitosan. From these data we suppose that the immobilized N-octadecylchitosan can form bilayer structures. Chemical Stability of N-OctadecylchitosanImmobilized Supports. Stability of N-octadecylchitosan-immobilized supports to alkali and acid was examined by IR spectroscopy. Any substantial change was not observed in spectra by the immersion in 0.5 N NaOH or 0.1 N HC1 overnight at ambient temperature. This chemical stability of N-octadecylchitosan-immobilized supports is attributable to the stability of the amide linkage used to the immobilization ofN-octadecylchitosan as well as the stability of the support material. For example, when chitosan is prepared from chitin, it is necessary to boil chitin in concentrated NaOH to hydrolyze the N-acetyl bond of GlcNAc. Elution from N-octadecylchitosan-immobilized supports was evaluated by measuring total organic carbon content in an eluent from a column packed with 20 mL ofthe supports. The column was filled with 0.5 N NaOH and allowed to stand overnight simulating the sterilization procedure in a chromatographic purification of pharmaceuticals. After NaOH was washed out, 50 mL of water was circulated in the column for 24 h. Total organic carbon in this water was 400 pgL. Even assuming that all of this organic carbon is due to eluted N-octadecylchitosan, the eluted N-octadecylchitosan (C, 65.7%) is calculated as 3.0 x g. This amount is 0.038% of the N-octadecylchitosan immobilized on the 20-mL supports.
Number of Immobilized Segments of N-Octadecylchitosan Molecule. As described in the introduction, morphology of immobilized polymeric amphiphiles might be influenced by a number of immobilized sites per one polymer molecule. Although a determination of the number of immobilized sites is not established, the following is speculated. Composition of chitosan used is assumed as 133 of GlcN segments and 27 of GlcNAc ones with a molecular weight of 2.67 x lo4. Molecular weight ofN-octadecylchitosan is calculated as 5.52 x lo4 (112 of GlcNC18 segments, 21 of GlcN, and 27 of GlcNAc) assuming that chitosan is N-octadecylated without depolymerization. WSC forms amide linkage from primary amino groups of GlcN and carboxyl groups ofthe supports. Ester linkages from hydroxyl groups of N-octadecylchitosan and carboxyl groups ofthe supports are possible. However, they are probably hydrolyzed by the treatment with 0.5 N NaOH performed after the immobilization reaction. Therefore the number of immobilized sites is not more than 21 per one N-octadecylchitosan molecule. The amount of N-octadecylchitosan immobilized to the 12.2 mL of carboxylated supports are 0.88pmol(4 mg/mL x 12.2 mL divided by 55 200). If WSC applied (35pmol) was all used to the immobilization, a number of immobilized segments per one polymer molecule is 40 (35 divided by 0.88). This is larger than the number of GlcN segments in an N-octadecylchitosan molecule, 21. WSC will be reacted with carboxyl groups to yield activated sites over the support surface. It is unlikely for all of the activated sites react with GlcN moieties of N-octadecylchitosan. We expect the number of immobilized sites per one polymer molecule is fewer than 21. Conclusion A novel methodology for the immobilization of lipid bilayer membrane is described. Polymeric bilayer membranes having some primary amino groups at hydrophilic moieties are covalentlyimmobilized to carboxylated porous supports by amidation in an aqueous phase using watersoluble carbodiimides. Although each lipid molecule is not directly bonded to the support material, all lipid molecules are covalently linked and immobilized to the support by a polymer main chain. This strategy and the mild reaction condition seem to result in the retention of the bilayer structure upon the immobilization procedure. The resulting porous supports bearing polymeric bilayer membranes possess superior chemical stability. They are expected to exhibit a superior selectivity to LPS compared to coventional hydrophobic adsorbents in the removal of pyrogenic LPS from protein solutions. LA950196U
