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Development of dialyzer with immobilized glycoconjugate polymers for removal of Shiga-toxin
ARTICLE IN PRESS
Biomaterials 27 (2006) 3304–3311 www.elsevier.com/locate/biomaterials
Development of dialyzer with immobilized glycoconjugate polymers for removal of Shiga-toxin Atsushi Miyagawaa, Miho Watanabeb,c, Katsura Igaib,d, Maria Carmelita Z. Kasuyaa, Yasuhiro Natorib, Kiyotaka Nishikawab,d, Kenichi Hatanakaa,e, a Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan Department of Clinical Pharmacology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655,Japan c Bioresources Research Laboratory, The Institute of Medical Chemistry, Hoshi University, 2-4-41, Ebara, Shinagawa-ku, Tokyo 142-8501, Japan d Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Japan e Center for Collaborative Research, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan
b
Received 5 October 2005; accepted 24 January 2006 Available online 24 February 2006
Abstract The dialyzer for Shiga-toxin elimination was developed and its performance was established. The dialyzer was prepared by immobilization of multivalent ligands. Glycoconjugate polymers having oligosaccharides and amino groups were synthesized to function as Shiga-toxin adsorbents. The amino group was utilized to immobilize the polymer inside the cellulose hollow fiber of the dialyzer. Cellulose hollow fibers packed in the dialyzer were carboxymethylated under moderate conditions. The glycoconjugate polymers were bound covalently to the hollow fibers of the dialyzer by condensation reaction between the amino group of the polymer and the carboxyl group of the cellulose hollow fiber. Shiga-toxin eliminabilities of the prepared dialyzers were evaluated at various conditions. Even at high concentration of protein such as FCS, the dialyzer showed an excellent performance for Shiga-toxin adsorption. r 2006 Elsevier Ltd. All rights reserved. Keywords: Adsorption; Dialyzer; Glycoconjugate polymer; Immobilization; Shiga-toxin
1. Introduction Infection with shigatoxigenic Escherichia coli (STEC) causes hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and neurological damages in humans [1]. Shiga-toxins (Stxs: Stx1, Stx2) produced by STEC are composed of one toxic subunit (A subunit) and five sugar recognizing subunits (B subunits). Since the A subunit (32 kDa) acts as an N-glycosidase that cleaves an adenine residue in 28S ribosomal RNA of a host cell, the cell is unable to synthesize proteins. The B subunits (7.7 kDa 5) recognize glycolipids on the cell surface and Stxs are incorporated into the cell. Stxs circulating in the blood bind to globotriaosylceramide (Gb3) on the cell surface of renal endothelial cells or red blood cells and then impair renal function [2–5]. The patients infected with STEC are Corresponding author. Tel.: +81 3 5452 6355; fax: +81 3 5452 6356.
E-mail address: [email protected] (K. Hatanaka). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.01.049
treated with antibiotics and/or dialysis. However, the application of antibiotics for the patient is not always effective in killing the bacteria that is probably releasing the toxins [6]. In the previous reports, two kinds of therapeutic agents which are carbosilane dendrimers with globotrioses [7] and acrylamide polymers containing globotrioses [8] were developed. Both agents were effective in neutralizing Stxs. The component of the later agent can be chemically changed. By adding a functionalizing monomer into the copolymerization system, the synthesized polymer acquires a new function. For example, adding a fluorescent monomer affords a polymer that exhibits fluorescence. In the previous work [9], glycoconjugate polymers which were functionalized by this method were synthesized. The polymers have sugar moieties, fluorescence labels and amino groups. The polymers were immobilized onto carboxymethylated cellulose membranes which can be used as affinity membranes for lectins.
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In this investigation, glycoconjugate polymers having globotrioses, fluorescence labels and amino groups were synthesized. The glycoconjugate polymers were immobilized onto cellulose hollow fibers of a dialyzer. The Shigatoxin adsorption ability of the dialyzer with immobilized polymers was investigated at various conditions. Furthermore, the inhibition of the polymers on the toxicity of Stxs was evaluated. 2. Materials and methods 2.1. Materials Unless otherwise stated, all commercially available solvents and reagents were used without further purification. Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,2-dichloroethane, dichloromethane, and pyridine were stored over molecular sieves 4 A˚. Methanol
(i) RnO
S
RnO
NH2-HCl
R1= globotriose (1)
R1= globotriose (4)
R2= lactose (2)
R2= lactose (5)
R3= cellobiose (3)
R3= cellobiose (6) (ii)
(iii) RnO
H C N O R1= globotriose (7) S
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was stored over molecular sieves 3 A˚. Tetrahydrofuran (THF) was stored over molecular sieves 5 A˚. Powdered molecular sieves were dried in vacuo at ca. 180 1C for at least 2 h. Acrylamide was recrystallized from benzene. Dialyzer (Asahi Hollow Fiber Dialyzer AM-UP-10) was purchased from Asahi Kasei Medical Co., Ltd. Stx1 and Stx2 were prepared according to methods described elsewhere [10].
2.2. General methods 1 H NMR spectra were recorded at 400 or 600 MHz using a Jeol JNM-AL400 or Jeol ECP-600 spectrometer in chloroform-d or deuterium oxide. 13C NMR spectra were recorded at 100.6 or 150.9 MHz with the same instruments. Tetramethylsilane (TMS) was used as the internal standard. Assignments in the NMR spectra were made by first-order analysis of spectra, and supported by correlation spectroscopy and heteronuclear chemical shift correlation. The average molecular weights of the polymers were estimated by size-exclusion chromatography (SEC) using a TOSOH TSKgel G-Oligo-PW column, TSKgel G2500PWXL column, TSKgel G3000PWXL column, and TSKgel G4000PWXL column with using pullulans (5.8, 12.2, 23.7, 48.0, 100, 186, 380 kDa, Shodex Standard P-82) as standards. Reactions were monitored by thin-layer chromatography (TLC) on a precoated plate of silica gel 60 F254 (layer thickness, 0.25 mm; E. Merk, Darmstadt, Germany). For detection of intermediates, TLC sheets were dipped in (a) a solution of 85:10:5 (v/v/v) methanol-p-anisaldehyde-concentrated sulfuric acid and heated for a few minutes (for carbohydrates), or (b) an aqueous solution of 5 wt% potassium permanganate and heated similarly (for double bond). Column chromatography was performed on silica gel (Silica Gel 60; 40–63 mm, E. Merck, or Silica Gel 60, spherical neutral; 40–100 mm, E. Merck).
2.3. Polymerization
R2=lactose (8) R3=cellobiose (9) N H N
O2S N H
C O
H N
H2N
(10)
C O
H2N C O
(11)
(12)
Fig. 1. Reagents and conditions: (i) HSCH2CH2NH2 HCl, MeOH, hn (254 nm), room temperature; (ii) CH2¼CHCOCl, Na2CO3, MeOH, 0 1C, then Ac2O, pyridine, room temperature; (iii) NaOMe, MeOH, room temperature.
The glycosyl monomers were prepared by the method described in Fig. 1. A solution of the glycosyl monomer 7 (globotriose), 8 (lactose) or 9 (cellobiose), fluorescent monomer 10, amine monomer 11, and acrylamide in deionized water or DMSO was degassed using a diaphragm pump, and TEMED (0.1 or 0.2 eq for glycosyl monomer) and APS (0.04 or 0.08 eq for glycosyl monomer) were added to the solution. The reaction mixture was continuously stirred overnight at room temperature. The resulting product was purified by reprecipitation with a mixed solution of methanol and ethanol and lyophilized to give a water-soluble copolymer as a powder. This procedure was performed for each of the monomer ratios in Table 1. G series of glycoconjugate polymers (G1, G2, G3, and G4) have globotriose units, L series of glycoconjugate polymers (L1 and L2)
Table 1 Results of copolymerization Polymer
G1 G2 G3 G4 L1 L2 C1 a
Monomer ratio Glycosyl monomer 7, 8 or 9
Fluorescence monomer 10
Amine monomer 11
Acryl amide 12
1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.05 — — — 0.05 — —
0.1 0.05 0.05 0.05 0.1 0.05 0.05
4 — 4 8 4 — —
(7) (7) (7) (7) (8) (8) (9)
Total field
Polymer compositiona
Sugar content (mol%)
Mwb (kDa)
63.7 89 84.9 99 80.7 93.1 82.8
1:0.05:0.05:4.4 1:0.13 1:0.03:5.0 1:0.05:9.5 1:0.06:0.11:4.0 1:0.08 1:0.04
67.8 97.0 66.6 51.2 61.5 97.6 98.8
64 248 644 749 320 302 400
The polymer composition was determined from the integration value of 1H NMR. Mws were estimated by the SEC method with the TOSOH TSKgel G-Oligo-PW column, TSKgel G2500PWXL column, TSKgel G3000PWXL column and TSKgel G4000PWXL column [pullulans (5.8, 12.2, 23.7, 48.0, 100, 186, and 380 kDa, Shodex Standard P-82) were used as standards]. b
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have lactose units, and the glycoconjugate polymer C1 has cellobiose units. Dialyzers with immobilized glycoconjugate polymers were named D-(polymer number). For example, D-G2 denotes dialyzer with immobilized G2.
2.4. Cells Vero cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS). Cells were seeded in 96-well plastic microplate for cytotoxicity assays.
2.5. Shiga-toxin adsorption evaluation The evaluation method was carried out as follows: (1) 4.0 mg/ml or 40 ng/ml Stx1 solution (prepared by adding Stx1 to 1% BSA–PBS or FCS) was run through the dialyzer; (2) the circulated solution was collected at predefined times (10, 30 min, 1, 2, 4 h); (3) the samples were diluted with medium; (4) subconfluent Vero cells were treated with the diluted samples; and (5) the remaining amount of Stx1 in the circulated solution was evaluated by comparing with the number of living cells treated with standard Stx1 solution using a WST-8 Cell Counting Kit (Wako Pure Industries).
2.6. Western blotting An aqueous solution of Stx1 was circulated through the dialyzer for 4 h. Then, the dialyzer was washed sufficiently with PBS, and 4 M MgCl2 solution was circulated through the dialyzer for 30 min. The solution was evaporated and run on 16% SDS-PAGE. The gel was soaked in transfer buffer and proteins in the gel were transferred to a PVDF membrane (ATTO Corp.). The membrane was incubated with 5% skim milk TBS for 1 h for blocking. Then, the membrane was washed with TBS-T (0.2% Tween-20-TBS) three times and soaked in anti-Stx1 rabbit IgG (kindly provided by K. Nishikawa)–1% BSA TBS-T solution for 1 h. Subsequently, the membrane was washed with TBS-T three times and was soaked in anti-rabbit HRP goat IgG (SouthernBiotech)1% BSA TBS-T solution for 1 h. After the membrane was washed with TBS-T three times and TBS three times, it was blotted with chemiluminescence reagent (PerkinElmer).
2.7. Inhibition assay on cytotoxicity Subconfluent Vero cells in a 96-well plate were treated with Stx1 (10 pg/ ml) in the absence or the presence of the desired amount of a given glycoconjugate polymer for 72 h. Relative cell number was determined using a WST-8 Cell Counting Kit (Wako Pure Industries).
2.8. Kinetic analysis of binding Stxs to immobilized glycoconjugate polymers Stx1 and Stx2 immobilized on glycoconjugate polymers were quantified by using a BIAcore system instrument (Pharmacia Biosensor, Uppsala, Sweden). The glycoconjugate polymer (10 mg/ml) was injected into the system to become immobilized on CM5 sensor chip. Various concentrations of Stx1 and Stx2 were injected (time 0) over the immobilized glycoconjugate polymer at a flow rate of 20 ml/min for at least 5 min to reach a plateau at 25 1C. The resonance unit is an arbitrary unit used by the BIAcore system. The resonance unit value obtained from the immobilized polymer having lactose was subtracted from the data obtained from the immobilized polymer having globotriose for correction of the background. The binding kinetics were analyzed by Scatchard plot using the software BIAEVALUATION 3.0.
3. Results and discussion 3.1. Synthesis of glycoconjugate polymers Glycosyl monomers 7, 8 and 9 were prepared by a similar procedure as described previously [11] (Fig. 1). Compounds 1, 2 and 3 were irradiated with a UV lamp (254 nm) to afford compounds 4, 5 and 6 which were subsequently N-acryloylated and acetylated. After purification, glycosyl monomers 7, 8 and 9 having acrylamide group were obtained by deacetylation. Compound 10 which has a fluorescent dansyl group and compound 11 which has an amino group were synthesized as described previously [9]. As shown in Table 1, the monomers were copolymerized with, or without acrylamide, to afford various types of polymers according to previous method [9,11]. 3.2. Immobilization of polymers onto the hollow fibers in dialyzer The same method as the immobilization onto the membrane filter [9] was adapted to immobilize the glycoconjugate polymer onto hollow fiber in the dialyzer. First, the hollow fiber was carboxymethylated with bromoacetic acid as follows: A mixture of 0.5 or 1.0 M NaOH and saturated NaCl solution was circulated inside the hollow fiber, followed by the addition of bromoacetic acid to the solution. The dialyzer was then washed with water until the washings become neutral. Next, the polymer was immobilized onto the hollow fiber as follows: 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride was circulated inside the carboxymethylated hollow fiber followed by circulation of the polymers of L1 (lactose+fluorescence copolymer) or G1 (globotriose+ fluorescence copolymer). After the reaction, the dialyzer was washed sufficiently with water. Luminescence of the hollow fiber under irradiation with a UV lamp showed the existence of fluorescence labeled L1 or G1 (Fig. 2). The results suggested that the glycoconjugate polymer was immobilized onto the hollow fiber. Other polymers of G2 (globotriose polymer), G3, G4 (globotriose copolymers), L2 (lactose polymer), and C1 (cellobiose polymer) were also immobilized onto the carboxymethylated hollow fiber in the dialyzer in a similar
Fig. 2. Luminescence of hollow fibers of the dialyzer with immobilized glycoconjugate polymers (G1 and L2) during irradiation with a UV lamp.
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Table 2 Polymers which were immobilized to dialyzers Polymer
Immobilized polymer (mg)
Sugar content (mmol)
D-G2 D-G3 D-G4 D-L2 D-C1
32.0 20.3 24.3 36.1 30.6
42.0 12.5 8.88 61.9 53.8
Fig. 3. Experimental equipment component.
manner. After the immobilization reaction, the circulated solution was concentrated and non-immobilized polymer was weighed. Consequently, the amount of immobilized polymer was calculated by subtracting non-immobilized amount from the total amount used. Each of the dialyzers prepared by this method had the glycoconjugate polymer. The results are summarized in Table 2. 3.3. The Shiga-toxin adsorption ability of dialyzer with immobilized polymers Initially, the dialyzer with immobilized polymer G2 (globotriose polymer) (D-G2) was used to determine the ability of Shiga-toxin adsorption. After 1% BSA–PBS() solution was circulated through D-G2 for 30 min to block the non-specific adsorption, 4 mg/ml Stx1–PBS() solution (Stx1: 2.8 nmol) was circulated through D-G2 at a flow rate of 10 ml/min (Fig. 3). However, this blocking did not completely inhibit the non-specific adsorption (data not shown). Consequently, Stx1 was directly added to 1% BSA–PBS() solution and satisfactorily blocked the nonspecific adsorption. The result of the evaluation is shown in Fig. 3. D-G2 was highly efficient to eliminate Stx1 from 1% BSA–PBS() solution (Fig. 4(c)). By circulating the solution for only 10 min, Stx1 concentration was diluted to one-millionth part of the starting concentration (4 mg/ ml). As anticipated, the non-modified dialyzer (no polymer attached) was unable to adsorb Stx1 (Fig. 4(b)). Then, it was confirmed whether the Shiga-toxin adsorption was sugar specific, or not. The dialyzers with immobilized polymers L2 (lactose polymer) and C1 (cellobiose polymer) (D-L2 and D-C1) were evaluated by the same procedure. Although D-L2 did not adsorb Stx1 specifically, a small amount of Stx1 was non-specifically adsorbed for 4 h (Fig. 4(d)). D-C1 hardly adsorbed Stx1 (Fig. 4(e)). Consequently, these results confirmed that the globotriose was specifically recognized by Stx1. By considering the adsorption ability of D-G2, the dialyzers with immobilized polymer G3 and G4 (globotriose copolymers) (D-G3 and D-G4) were anticipated to absorb Stx1 efficiently. However, D-G3 and D-G4 did not show the adsorption ability for Stx1 (Fig. 4(f) and (g)). It was presumed that glycoconjugate polymers, such as G3 and G4 which are the copolymers with acrylamide, exhibit inhibitory effects as mentioned in previous report
[8] and other literature [12]. When these polymers were utilized by immobilizing onto a material, it can be suggested that the interaction between the polymers and materials involves noncovalent bonds, such as hydrogen bonds and hydrophobic interactions. In this case, the cellulose dialyzer and the glycoconjugate polymers have hydroxy groups, and the glycoconjugate polymers have amide groups. G3 and G4 have an acrylamide (–CONH2). In other words, it can be thought that an interaction occurs between the amido group of acrylamide and cellulose hollow fiber. Through this interaction, the polymer strongly adheres to the cellulose hollow fiber and thus, D-G3 and D-G4 did not show any adsorption to Stx1. Consequently, the polymer which was immobilized in a dialyzer needs for Shiga-toxin adsorption to have a degree of freedom. The result of further research by using BIAcore system and biological activities is shown later. D-G2 (dialyzer; globotriose polymer) was chosen for further investigation of Stx1 adsorption. The ability of D-G2 to eliminate Stx1 was evaluated at the circulation rate of 120 ml/min which is similar to an actual artificial dialysis. By changing to the faster circulation rate, it was necessary for adsorbing Stx1 to circulate for longer time (Fig. 4(h)). D-G2 had an enough ability to adsorb Stx1 for 4 h and could dilute the Stx1 concentration to onemillionth part of the starting concentration (4 mg/ml). Subsequently, the FCS was used as circulated solution. FCS contains more protein than 1% BSA–PBS solution. The evaluation for D-G2 in FCS is more similar to the evaluation in blood for evaluating the specific adsorption to Stx1. The circulation rate (120 ml/min) is almost the same as in artificial dialysis. The result showed that D-G2 could specifically and efficiently adsorb Stx1 in FCS (Fig. 5(b)). A 7% protein concentration in FCS did not affect the adsorption ability of D-G2 at all. It was found that the polymer G2 (globotriose polymer) which was immobilized to dialyzer highly recognized Stx1. Comparing with concentration of Stxs in patient’s blood, the experimental concentration of Stx1 was high. Therefore, the concentration of Stx1 was changed to 40 ng/ml (Stx1: 28 pmol). Dilute Stx1 solution was circulated in D-G2 and
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3308 Standard
Control
120 10 min 30 min 1h 2h 4h
100 80
Cell viability (%)
Cell viability (%)
100 80 60 40
60 40
20
20
0 0.00001 0.0001 0.001
0.01
0.1
1
10
100
Stx1 concentration (ng/ml)
(a)
0 0.001
1000 (b)
D-G2
120
D-L2 100
80 60 10 min 30 min 1h 2h 4h
40 20
(c)
10 min 30 min 1h 2h 4h
80
0.01
0.1
1
10
100
Cell viability (%)
Cell viability (%)
100
0 0.001
10 0.01 0.1 1 100 1000 Stx1 concentration of circulated solution by dilution (ng/ml)
60 40 20 0 -20 0.001
1000 (d)
Stx1 concentration of circulated solution by dilution (ng/ml)
0.01
0.1
Cell viability (%)
100
1000
80 60 40
10 min 30 min 1h 2h 4h
100 80 Cell viability (%)
10 min 30 min 1h 2h 4h
100
60 40 20
20
0.01
0.1
1
10
100
0 0.001
1000
Stx1 concentration of circulated solution by dilution (ng/ml)
(e)
10
D-G3
D-C1
0 0.001
1
Stx1 concentration of circulated solution by dilution (ng/ml)
0.01
0.1
1
10
100
1000
Stx1 concentration of circulated solution by dilution (ng/ml)
(f)
D-G2
D-G4 120
Cell viability (%)
80 60 40
100 Cell viability (%)
10 min 30 min 1h 2h 4h
100
(g)
60 10 min 30 min 1h 2h 4h
40 20
20 0 0.001
80
0.01
0.1
1
10
100
Stx1 concentration of circulated solution by dilution (ng/ml)
0 0.001
1000 (h)
0.01
0.1
1
10
100
1000
Stx1 concentration of circulated solution by dilution (ng/ml)
Fig. 4. The evaluation of the Stx1 adsorption ability by dialyzers. (a) Standard toxic solution (mean7SE, n ¼ 8). (b) Control (no polymer attached) (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, mean7SE, n ¼ 3) (c) D-G2 (condition; Stx1 concentration 4 mg/ ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, mean7SE, n ¼ 4). (d) D-L2 (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, n ¼ 2). (e) D-C1 (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, n ¼ 2). (f) D-G3 (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, n ¼ 2). (g) D-G4 (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, n ¼ 2). (h) D-G2 (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 120 ml/min, n ¼ 1). The data are presented as percentage of cell viability in the absence of Stx1.
ARTICLE IN PRESS A. Miyagawa et al. / Biomaterials 27 (2006) 3304–3311 Control
100 80 60 40
100
20
Cell viability (%)
(c)
D-G2
80 60 10min 30min 1h 2h 4h
0 0.0001
10 min 30 min 1h 2h 4h
40
120
100
20
60
0 0.001 0.01 0.1 1 10 100 1000 Stx1 concentration of circulated solution by dilution (b) (ng/ml)
Control
120
40
80
20
0 0.001 0.01 0.1 1 10 100 1000 Stx1 concentration of circulated solution by dilution (ng/ml)
Cell viability (%)
(a)
D-G2
120 10 min 30 min 1h 2h 4h
Cell viability (%)
Cell viability (%)
120
0.001
3309
100 80 60 10 min 30 min 1h 2h 4h
40 20
0.01
0.1
1
0 0.0001
10
Stx1 concentration of circulated solution by dilution (ng/ml)
(d)
0.001
0.01
0.1
1
10
Stx1 concentration of circulated solution by dilution (ng/ml)
Fig. 5. The evaluation of the Stx1 adsorption ability by Dialyzers. (a) Control (no polymer attached) (condition; Stx1 concentration 4 mg/ml, solvent FCS, circulation rate 120 ml/min, mean7SE, n ¼ 3) (b) D-G2 (condition; Stx1 concentration 4 mg/ml, solvent FCS, circulation rate 120 ml/min, mean7SE, n ¼ 3). (c) Control (no polymer attached) (condition; Stx1 concentration 40 ng/ml, solvent FCS, circulation rate 120 ml/min, mean7SE, n ¼ 3). (d) D-G2 (condition; Stx1 concentration 40 ng/ml, solvent FCS, circulation rate 120 ml/min, mean7SE, n ¼ 3). The data are presented as the percentage of cell viability in the absence of Stx1.
the Stx1 concentration in the circulated solution was measured. The result of the control experiment showed that there was non-specific adsorption to FCS and/or dialyzer, but the circulated solution was still toxic (Fig. 5(c)). On the other hand, the result of D-G2 (Fig. 5(d)) showed the specific elimination. By circulating the solution for only 10 min, D-G2 mostly eliminated Stx1 even from the dilute Stx1 solution. This result showed that D-G2 had high specificity for Stx1 adsorption, and the protein in FCS was hardly adsorbed to immobilize G2. Consequently, dialyzer D-G2 has high potency of adsorbing Stxs in blood. 3.4. Western blotting for ascertaining adsorption of Stx1 Adsorption of Stx1 to the dialyzer and its specificity for a glycoconjugate polymer was ascertained. After Stx1–1% BSA–PBS solutions were circulated through D-G2 (dialyzer; globotriose polymer) and D-L2 (dialyzer; lactose polymer) for 4 h, D-G2 and D-L2 was washed sufficiently with PBS. Adsorbed Stx1 was eluted with 4 M MgCl2 solution from D-G2 and D-L2. The eluted solution was adapted to Western blotting and the result is shown in Fig. 6. The film obviously indicated a specific adsorption of Stx1 on the dialyzer. Stx1-based A and B subunit bands appeared only at the D-G2 lane. Consequently, Stx1
was caught by a globotrioses of G2 instead of acrylamide main chain. 3.5. Inhibition of the biological activities of Stxs by polymers All of the polymers were examined for the inhibition of cytotoxicity of Stxs (Fig. 7(a) and (b)). G2 (globotriose polymer) markedly inhibited the cytotoxic activity of Stxs. However, G3 and G4 (globotriose copolymers), as well as L2 (lactose polymer) have only low activity. C1 (cellobiose polymer) had no inhibitory effect for Stxs at any concentration. This result corresponded with the evaluations of the Stx1 adsorption abilities of D-G3, D-G4 (dialyzers; globotriose copolymers), and D-L2 (dialyzer; lactose polymer). Consequently, the reason that G3 and G4 did not have the inhibitory activity can be the same as the reason why D-G3 and D-G4 did not have the adsorption ability. The reason for this is that the glycoconjugate polymer without acrylamide, G2, and with acrylamide, G3 and G4 (globotriose copolymers), may each have a different conformation in aqueous solution. Furthermore, in order to prepare a dialyzer which can eliminate Stxs efficiently, it is necessary that a glycoconjugate polymer immobilized to a dialyzer has a high biological activity like G2.
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3.6. Kd of Stxs for polymer G2 by using BIAcore system These polymers G2 (globotriose polymer) and L2 (lactose polymer) were immobilized on a BIAcore chip. The surface of a sensor chip coated with CM-dextran (CM5) is similar to that of the cellulose hollow fibers except for the hydrophilicity. The Kd values were determined by Scatchard plot analysis. Stx1 and Stx2 had very low Kd (16.476.5 and 13.571.2 nmol/L) for G2. Consequently, Stx1 had a Kd value, which was the expected result of the dialyzer evaluation of D-G2 (dialyzer; globotriose polymer), for G2. The Kd value of Stx2 for G2 is anticipated and D-G2 also has the high ability of Stx2 adsorption. 4. Conclusion
Fig. 6. The evidence of the Stx1 adsorption for the dialyzers using Western blotting. (a) Standard. (b) The eluted solution from D-L2. (c) The eluted solution from D-G2. Stx1
120
Cell viability (%)
100 80
G2 G3 G4 L2 C1
We developed a Shiga-toxin elimination dialyzer which has immobilized glycoconjugate polymer containing globotriose. D-G2 (dialyzer; globotriose polymer) efficiently adsorbed Stx1 from both dilute and concentrated Stx1 solutions. Moreover, the dialyzer eliminated Stx1 from FCS solution, which has a high protein concentration. The inhibitory effects of the polymers on the cytotoxicity of Stx1 and Stx2 were also determined. G2 (globotriose polymer) showed high inhibitory effect on both Stx1 and Stx2. Kd values of G2, which were measured by BIAcore system, were in the nM range for Stx1 and Stx2. G2 showed appreciably favorable results for each of the evaluations. Significantly, D-G2 showed potential to eliminate Stx1 and Stx2 from blood.
60
References 40 20 0 0.000001 0.00001 0.0001
(a)
Cell viability (%)
60
1
10
100
1
10
100
Stx2
100
80
0.001 0.01 0.1 Sugar content (µM)
G2 G3 G4 L2 C1
40
20
0 0.000001 0.00001 0.0001
(b)
0.001 0.01 0.1 Sugar content (µΜ)
Fig. 7. Cytotoxicity assay. (a) using Stx1 (mean7SE, n ¼ 3). (b) using Stx2 (mean7SE, n ¼ 3). The data are presented as percentage of cell viability in the absence of Stx1.
[1] Lingwood CA. Role of verotoxin receptors in pathogenesis. Trends Microbiol 1996;4:147–53. [2] Pozsgay V, Trinh L, Shiloach J, Robbins JB, Donohue-Rolfe A, Calderwood SB. Purification of subunit B of Shiga toxin using a synthetic trisaccharide-based affinity matrix. Bioconjugate Chem 1996;7:45–55. [3] Mu¨ller D, Vic G, Critchley P, Crout DHG, Lea N, Roberts L, et al. Chemical synthesis of globotriose and galabiose: relative stabilities of their complexes with Escherichia coli Shiga-like toxin-1 as determined by denaturation-titration with guanidium chloride. J Chem Soc Perkin Trans 1998;1:2287–94. [4] Hagnerelle X, Plisson C, Lambert O, Macro S, Rigaud JL, Le´vy D. Two-dimensional structures of Shiga toxin B-subunit and of a chimera bound to glycolipid receptor Gb3. J Struct Biol 2002;139:113–21. [5] Green DA, Murphy WG, Uttley WS. Haemolytic uraemic syndrome: prognostic factors. Clin Lab Haem 2000;22:11–4. [6] Ling H, Pannu NS, Boodhoo A, Armstrong GD, Clark CG, Brunton JL, et al. A mutant Shiga-like toxin IIe bound to its receptor Gb3: structure of a group II Shiga-like toxin with altered binding specificity. Structure 2000;8:253–64. [7] Nishikawa K, Matsuoka K, Kita E, Okabe N, Mizuguchi M, Hino K, et al. A therapeutic agent with oriented carbohydrates for treatment of infections by Shiga toxin-producing Escherichia coli O157:H7. Proc Natl Acad Sci 2002;99:7669–74. [8] Watanabe M, Matsuoka K, Kita E, Igai K, Higashi N, Miyagawa A, et al. Oral therapeutic agents with highly clustered globotriose for
ARTICLE IN PRESS A. Miyagawa et al. / Biomaterials 27 (2006) 3304–3311 treatment of Shiga toxigenic Escherichia coli infection. J Infect Dis 2004;189:360–8. [9] Miyagawa A, Kasuya MC, Hatanaka K. Immobilization of glycoconjugate polymers on cellulose membrane for affinity separation. Bull Chem Soc Jpn 2006;79:348–56. [10] Noda M, Yutsudo T, Nakabayashi N, Hirayama T, Takeda Y. Purification and some properties of Shiga-like toxin from Escherichia coli O157: H7 that is immunologically identical to Shiga toxin. Microb Pathog 1987;2:339–49.
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[11] Miyagawa A, Kurosawa H, Watanabe T, Koyama T, Terunuma D, Matsuoka K. Synthesis of glycoconjugate polymer carrying globotriaose as artificial multivalent ligand for Shiga toxinproducing Escherichia coli O157: H7. Carbohydr Res 2004;57: 441–50. [12] Dohi H, Nishida Y, Mizuno M, Shinkai M, Kobayashi T, Takeda T, et al. Synthesis of an artificial glycoconjugate polymer carrying Pk-antigenic trisaccharide and its potent neutralization activity against Shiga-like toxin. Bioorg Med Chem 1999;7:2053–62.
Biomaterials 27 (2006) 3304–3311 www.elsevier.com/locate/biomaterials
Development of dialyzer with immobilized glycoconjugate polymers for removal of Shiga-toxin Atsushi Miyagawaa, Miho Watanabeb,c, Katsura Igaib,d, Maria Carmelita Z. Kasuyaa, Yasuhiro Natorib, Kiyotaka Nishikawab,d, Kenichi Hatanakaa,e, a Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan Department of Clinical Pharmacology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655,Japan c Bioresources Research Laboratory, The Institute of Medical Chemistry, Hoshi University, 2-4-41, Ebara, Shinagawa-ku, Tokyo 142-8501, Japan d Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Japan e Center for Collaborative Research, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan
b
Received 5 October 2005; accepted 24 January 2006 Available online 24 February 2006
Abstract The dialyzer for Shiga-toxin elimination was developed and its performance was established. The dialyzer was prepared by immobilization of multivalent ligands. Glycoconjugate polymers having oligosaccharides and amino groups were synthesized to function as Shiga-toxin adsorbents. The amino group was utilized to immobilize the polymer inside the cellulose hollow fiber of the dialyzer. Cellulose hollow fibers packed in the dialyzer were carboxymethylated under moderate conditions. The glycoconjugate polymers were bound covalently to the hollow fibers of the dialyzer by condensation reaction between the amino group of the polymer and the carboxyl group of the cellulose hollow fiber. Shiga-toxin eliminabilities of the prepared dialyzers were evaluated at various conditions. Even at high concentration of protein such as FCS, the dialyzer showed an excellent performance for Shiga-toxin adsorption. r 2006 Elsevier Ltd. All rights reserved. Keywords: Adsorption; Dialyzer; Glycoconjugate polymer; Immobilization; Shiga-toxin
1. Introduction Infection with shigatoxigenic Escherichia coli (STEC) causes hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and neurological damages in humans [1]. Shiga-toxins (Stxs: Stx1, Stx2) produced by STEC are composed of one toxic subunit (A subunit) and five sugar recognizing subunits (B subunits). Since the A subunit (32 kDa) acts as an N-glycosidase that cleaves an adenine residue in 28S ribosomal RNA of a host cell, the cell is unable to synthesize proteins. The B subunits (7.7 kDa 5) recognize glycolipids on the cell surface and Stxs are incorporated into the cell. Stxs circulating in the blood bind to globotriaosylceramide (Gb3) on the cell surface of renal endothelial cells or red blood cells and then impair renal function [2–5]. The patients infected with STEC are Corresponding author. Tel.: +81 3 5452 6355; fax: +81 3 5452 6356.
E-mail address: [email protected] (K. Hatanaka). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.01.049
treated with antibiotics and/or dialysis. However, the application of antibiotics for the patient is not always effective in killing the bacteria that is probably releasing the toxins [6]. In the previous reports, two kinds of therapeutic agents which are carbosilane dendrimers with globotrioses [7] and acrylamide polymers containing globotrioses [8] were developed. Both agents were effective in neutralizing Stxs. The component of the later agent can be chemically changed. By adding a functionalizing monomer into the copolymerization system, the synthesized polymer acquires a new function. For example, adding a fluorescent monomer affords a polymer that exhibits fluorescence. In the previous work [9], glycoconjugate polymers which were functionalized by this method were synthesized. The polymers have sugar moieties, fluorescence labels and amino groups. The polymers were immobilized onto carboxymethylated cellulose membranes which can be used as affinity membranes for lectins.
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In this investigation, glycoconjugate polymers having globotrioses, fluorescence labels and amino groups were synthesized. The glycoconjugate polymers were immobilized onto cellulose hollow fibers of a dialyzer. The Shigatoxin adsorption ability of the dialyzer with immobilized polymers was investigated at various conditions. Furthermore, the inhibition of the polymers on the toxicity of Stxs was evaluated. 2. Materials and methods 2.1. Materials Unless otherwise stated, all commercially available solvents and reagents were used without further purification. Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,2-dichloroethane, dichloromethane, and pyridine were stored over molecular sieves 4 A˚. Methanol
(i) RnO
S
RnO
NH2-HCl
R1= globotriose (1)
R1= globotriose (4)
R2= lactose (2)
R2= lactose (5)
R3= cellobiose (3)
R3= cellobiose (6) (ii)
(iii) RnO
H C N O R1= globotriose (7) S
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was stored over molecular sieves 3 A˚. Tetrahydrofuran (THF) was stored over molecular sieves 5 A˚. Powdered molecular sieves were dried in vacuo at ca. 180 1C for at least 2 h. Acrylamide was recrystallized from benzene. Dialyzer (Asahi Hollow Fiber Dialyzer AM-UP-10) was purchased from Asahi Kasei Medical Co., Ltd. Stx1 and Stx2 were prepared according to methods described elsewhere [10].
2.2. General methods 1 H NMR spectra were recorded at 400 or 600 MHz using a Jeol JNM-AL400 or Jeol ECP-600 spectrometer in chloroform-d or deuterium oxide. 13C NMR spectra were recorded at 100.6 or 150.9 MHz with the same instruments. Tetramethylsilane (TMS) was used as the internal standard. Assignments in the NMR spectra were made by first-order analysis of spectra, and supported by correlation spectroscopy and heteronuclear chemical shift correlation. The average molecular weights of the polymers were estimated by size-exclusion chromatography (SEC) using a TOSOH TSKgel G-Oligo-PW column, TSKgel G2500PWXL column, TSKgel G3000PWXL column, and TSKgel G4000PWXL column with using pullulans (5.8, 12.2, 23.7, 48.0, 100, 186, 380 kDa, Shodex Standard P-82) as standards. Reactions were monitored by thin-layer chromatography (TLC) on a precoated plate of silica gel 60 F254 (layer thickness, 0.25 mm; E. Merk, Darmstadt, Germany). For detection of intermediates, TLC sheets were dipped in (a) a solution of 85:10:5 (v/v/v) methanol-p-anisaldehyde-concentrated sulfuric acid and heated for a few minutes (for carbohydrates), or (b) an aqueous solution of 5 wt% potassium permanganate and heated similarly (for double bond). Column chromatography was performed on silica gel (Silica Gel 60; 40–63 mm, E. Merck, or Silica Gel 60, spherical neutral; 40–100 mm, E. Merck).
2.3. Polymerization
R2=lactose (8) R3=cellobiose (9) N H N
O2S N H
C O
H N
H2N
(10)
C O
H2N C O
(11)
(12)
Fig. 1. Reagents and conditions: (i) HSCH2CH2NH2 HCl, MeOH, hn (254 nm), room temperature; (ii) CH2¼CHCOCl, Na2CO3, MeOH, 0 1C, then Ac2O, pyridine, room temperature; (iii) NaOMe, MeOH, room temperature.
The glycosyl monomers were prepared by the method described in Fig. 1. A solution of the glycosyl monomer 7 (globotriose), 8 (lactose) or 9 (cellobiose), fluorescent monomer 10, amine monomer 11, and acrylamide in deionized water or DMSO was degassed using a diaphragm pump, and TEMED (0.1 or 0.2 eq for glycosyl monomer) and APS (0.04 or 0.08 eq for glycosyl monomer) were added to the solution. The reaction mixture was continuously stirred overnight at room temperature. The resulting product was purified by reprecipitation with a mixed solution of methanol and ethanol and lyophilized to give a water-soluble copolymer as a powder. This procedure was performed for each of the monomer ratios in Table 1. G series of glycoconjugate polymers (G1, G2, G3, and G4) have globotriose units, L series of glycoconjugate polymers (L1 and L2)
Table 1 Results of copolymerization Polymer
G1 G2 G3 G4 L1 L2 C1 a
Monomer ratio Glycosyl monomer 7, 8 or 9
Fluorescence monomer 10
Amine monomer 11
Acryl amide 12
1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.05 — — — 0.05 — —
0.1 0.05 0.05 0.05 0.1 0.05 0.05
4 — 4 8 4 — —
(7) (7) (7) (7) (8) (8) (9)
Total field
Polymer compositiona
Sugar content (mol%)
Mwb (kDa)
63.7 89 84.9 99 80.7 93.1 82.8
1:0.05:0.05:4.4 1:0.13 1:0.03:5.0 1:0.05:9.5 1:0.06:0.11:4.0 1:0.08 1:0.04
67.8 97.0 66.6 51.2 61.5 97.6 98.8
64 248 644 749 320 302 400
The polymer composition was determined from the integration value of 1H NMR. Mws were estimated by the SEC method with the TOSOH TSKgel G-Oligo-PW column, TSKgel G2500PWXL column, TSKgel G3000PWXL column and TSKgel G4000PWXL column [pullulans (5.8, 12.2, 23.7, 48.0, 100, 186, and 380 kDa, Shodex Standard P-82) were used as standards]. b
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have lactose units, and the glycoconjugate polymer C1 has cellobiose units. Dialyzers with immobilized glycoconjugate polymers were named D-(polymer number). For example, D-G2 denotes dialyzer with immobilized G2.
2.4. Cells Vero cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS). Cells were seeded in 96-well plastic microplate for cytotoxicity assays.
2.5. Shiga-toxin adsorption evaluation The evaluation method was carried out as follows: (1) 4.0 mg/ml or 40 ng/ml Stx1 solution (prepared by adding Stx1 to 1% BSA–PBS or FCS) was run through the dialyzer; (2) the circulated solution was collected at predefined times (10, 30 min, 1, 2, 4 h); (3) the samples were diluted with medium; (4) subconfluent Vero cells were treated with the diluted samples; and (5) the remaining amount of Stx1 in the circulated solution was evaluated by comparing with the number of living cells treated with standard Stx1 solution using a WST-8 Cell Counting Kit (Wako Pure Industries).
2.6. Western blotting An aqueous solution of Stx1 was circulated through the dialyzer for 4 h. Then, the dialyzer was washed sufficiently with PBS, and 4 M MgCl2 solution was circulated through the dialyzer for 30 min. The solution was evaporated and run on 16% SDS-PAGE. The gel was soaked in transfer buffer and proteins in the gel were transferred to a PVDF membrane (ATTO Corp.). The membrane was incubated with 5% skim milk TBS for 1 h for blocking. Then, the membrane was washed with TBS-T (0.2% Tween-20-TBS) three times and soaked in anti-Stx1 rabbit IgG (kindly provided by K. Nishikawa)–1% BSA TBS-T solution for 1 h. Subsequently, the membrane was washed with TBS-T three times and was soaked in anti-rabbit HRP goat IgG (SouthernBiotech)1% BSA TBS-T solution for 1 h. After the membrane was washed with TBS-T three times and TBS three times, it was blotted with chemiluminescence reagent (PerkinElmer).
2.7. Inhibition assay on cytotoxicity Subconfluent Vero cells in a 96-well plate were treated with Stx1 (10 pg/ ml) in the absence or the presence of the desired amount of a given glycoconjugate polymer for 72 h. Relative cell number was determined using a WST-8 Cell Counting Kit (Wako Pure Industries).
2.8. Kinetic analysis of binding Stxs to immobilized glycoconjugate polymers Stx1 and Stx2 immobilized on glycoconjugate polymers were quantified by using a BIAcore system instrument (Pharmacia Biosensor, Uppsala, Sweden). The glycoconjugate polymer (10 mg/ml) was injected into the system to become immobilized on CM5 sensor chip. Various concentrations of Stx1 and Stx2 were injected (time 0) over the immobilized glycoconjugate polymer at a flow rate of 20 ml/min for at least 5 min to reach a plateau at 25 1C. The resonance unit is an arbitrary unit used by the BIAcore system. The resonance unit value obtained from the immobilized polymer having lactose was subtracted from the data obtained from the immobilized polymer having globotriose for correction of the background. The binding kinetics were analyzed by Scatchard plot using the software BIAEVALUATION 3.0.
3. Results and discussion 3.1. Synthesis of glycoconjugate polymers Glycosyl monomers 7, 8 and 9 were prepared by a similar procedure as described previously [11] (Fig. 1). Compounds 1, 2 and 3 were irradiated with a UV lamp (254 nm) to afford compounds 4, 5 and 6 which were subsequently N-acryloylated and acetylated. After purification, glycosyl monomers 7, 8 and 9 having acrylamide group were obtained by deacetylation. Compound 10 which has a fluorescent dansyl group and compound 11 which has an amino group were synthesized as described previously [9]. As shown in Table 1, the monomers were copolymerized with, or without acrylamide, to afford various types of polymers according to previous method [9,11]. 3.2. Immobilization of polymers onto the hollow fibers in dialyzer The same method as the immobilization onto the membrane filter [9] was adapted to immobilize the glycoconjugate polymer onto hollow fiber in the dialyzer. First, the hollow fiber was carboxymethylated with bromoacetic acid as follows: A mixture of 0.5 or 1.0 M NaOH and saturated NaCl solution was circulated inside the hollow fiber, followed by the addition of bromoacetic acid to the solution. The dialyzer was then washed with water until the washings become neutral. Next, the polymer was immobilized onto the hollow fiber as follows: 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride was circulated inside the carboxymethylated hollow fiber followed by circulation of the polymers of L1 (lactose+fluorescence copolymer) or G1 (globotriose+ fluorescence copolymer). After the reaction, the dialyzer was washed sufficiently with water. Luminescence of the hollow fiber under irradiation with a UV lamp showed the existence of fluorescence labeled L1 or G1 (Fig. 2). The results suggested that the glycoconjugate polymer was immobilized onto the hollow fiber. Other polymers of G2 (globotriose polymer), G3, G4 (globotriose copolymers), L2 (lactose polymer), and C1 (cellobiose polymer) were also immobilized onto the carboxymethylated hollow fiber in the dialyzer in a similar
Fig. 2. Luminescence of hollow fibers of the dialyzer with immobilized glycoconjugate polymers (G1 and L2) during irradiation with a UV lamp.
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Table 2 Polymers which were immobilized to dialyzers Polymer
Immobilized polymer (mg)
Sugar content (mmol)
D-G2 D-G3 D-G4 D-L2 D-C1
32.0 20.3 24.3 36.1 30.6
42.0 12.5 8.88 61.9 53.8
Fig. 3. Experimental equipment component.
manner. After the immobilization reaction, the circulated solution was concentrated and non-immobilized polymer was weighed. Consequently, the amount of immobilized polymer was calculated by subtracting non-immobilized amount from the total amount used. Each of the dialyzers prepared by this method had the glycoconjugate polymer. The results are summarized in Table 2. 3.3. The Shiga-toxin adsorption ability of dialyzer with immobilized polymers Initially, the dialyzer with immobilized polymer G2 (globotriose polymer) (D-G2) was used to determine the ability of Shiga-toxin adsorption. After 1% BSA–PBS() solution was circulated through D-G2 for 30 min to block the non-specific adsorption, 4 mg/ml Stx1–PBS() solution (Stx1: 2.8 nmol) was circulated through D-G2 at a flow rate of 10 ml/min (Fig. 3). However, this blocking did not completely inhibit the non-specific adsorption (data not shown). Consequently, Stx1 was directly added to 1% BSA–PBS() solution and satisfactorily blocked the nonspecific adsorption. The result of the evaluation is shown in Fig. 3. D-G2 was highly efficient to eliminate Stx1 from 1% BSA–PBS() solution (Fig. 4(c)). By circulating the solution for only 10 min, Stx1 concentration was diluted to one-millionth part of the starting concentration (4 mg/ ml). As anticipated, the non-modified dialyzer (no polymer attached) was unable to adsorb Stx1 (Fig. 4(b)). Then, it was confirmed whether the Shiga-toxin adsorption was sugar specific, or not. The dialyzers with immobilized polymers L2 (lactose polymer) and C1 (cellobiose polymer) (D-L2 and D-C1) were evaluated by the same procedure. Although D-L2 did not adsorb Stx1 specifically, a small amount of Stx1 was non-specifically adsorbed for 4 h (Fig. 4(d)). D-C1 hardly adsorbed Stx1 (Fig. 4(e)). Consequently, these results confirmed that the globotriose was specifically recognized by Stx1. By considering the adsorption ability of D-G2, the dialyzers with immobilized polymer G3 and G4 (globotriose copolymers) (D-G3 and D-G4) were anticipated to absorb Stx1 efficiently. However, D-G3 and D-G4 did not show the adsorption ability for Stx1 (Fig. 4(f) and (g)). It was presumed that glycoconjugate polymers, such as G3 and G4 which are the copolymers with acrylamide, exhibit inhibitory effects as mentioned in previous report
[8] and other literature [12]. When these polymers were utilized by immobilizing onto a material, it can be suggested that the interaction between the polymers and materials involves noncovalent bonds, such as hydrogen bonds and hydrophobic interactions. In this case, the cellulose dialyzer and the glycoconjugate polymers have hydroxy groups, and the glycoconjugate polymers have amide groups. G3 and G4 have an acrylamide (–CONH2). In other words, it can be thought that an interaction occurs between the amido group of acrylamide and cellulose hollow fiber. Through this interaction, the polymer strongly adheres to the cellulose hollow fiber and thus, D-G3 and D-G4 did not show any adsorption to Stx1. Consequently, the polymer which was immobilized in a dialyzer needs for Shiga-toxin adsorption to have a degree of freedom. The result of further research by using BIAcore system and biological activities is shown later. D-G2 (dialyzer; globotriose polymer) was chosen for further investigation of Stx1 adsorption. The ability of D-G2 to eliminate Stx1 was evaluated at the circulation rate of 120 ml/min which is similar to an actual artificial dialysis. By changing to the faster circulation rate, it was necessary for adsorbing Stx1 to circulate for longer time (Fig. 4(h)). D-G2 had an enough ability to adsorb Stx1 for 4 h and could dilute the Stx1 concentration to onemillionth part of the starting concentration (4 mg/ml). Subsequently, the FCS was used as circulated solution. FCS contains more protein than 1% BSA–PBS solution. The evaluation for D-G2 in FCS is more similar to the evaluation in blood for evaluating the specific adsorption to Stx1. The circulation rate (120 ml/min) is almost the same as in artificial dialysis. The result showed that D-G2 could specifically and efficiently adsorb Stx1 in FCS (Fig. 5(b)). A 7% protein concentration in FCS did not affect the adsorption ability of D-G2 at all. It was found that the polymer G2 (globotriose polymer) which was immobilized to dialyzer highly recognized Stx1. Comparing with concentration of Stxs in patient’s blood, the experimental concentration of Stx1 was high. Therefore, the concentration of Stx1 was changed to 40 ng/ml (Stx1: 28 pmol). Dilute Stx1 solution was circulated in D-G2 and
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3308 Standard
Control
120 10 min 30 min 1h 2h 4h
100 80
Cell viability (%)
Cell viability (%)
100 80 60 40
60 40
20
20
0 0.00001 0.0001 0.001
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1
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(a)
0 0.001
1000 (b)
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D-L2 100
80 60 10 min 30 min 1h 2h 4h
40 20
(c)
10 min 30 min 1h 2h 4h
80
0.01
0.1
1
10
100
Cell viability (%)
Cell viability (%)
100
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60 40 20 0 -20 0.001
1000 (d)
Stx1 concentration of circulated solution by dilution (ng/ml)
0.01
0.1
Cell viability (%)
100
1000
80 60 40
10 min 30 min 1h 2h 4h
100 80 Cell viability (%)
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60 40 20
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(e)
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0.01
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1000
Stx1 concentration of circulated solution by dilution (ng/ml)
(f)
D-G2
D-G4 120
Cell viability (%)
80 60 40
100 Cell viability (%)
10 min 30 min 1h 2h 4h
100
(g)
60 10 min 30 min 1h 2h 4h
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20 0 0.001
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0 0.001
1000 (h)
0.01
0.1
1
10
100
1000
Stx1 concentration of circulated solution by dilution (ng/ml)
Fig. 4. The evaluation of the Stx1 adsorption ability by dialyzers. (a) Standard toxic solution (mean7SE, n ¼ 8). (b) Control (no polymer attached) (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, mean7SE, n ¼ 3) (c) D-G2 (condition; Stx1 concentration 4 mg/ ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, mean7SE, n ¼ 4). (d) D-L2 (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, n ¼ 2). (e) D-C1 (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, n ¼ 2). (f) D-G3 (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, n ¼ 2). (g) D-G4 (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 10 ml/min, n ¼ 2). (h) D-G2 (condition; Stx1 concentration 4 mg/ml, solvent 1% BSA–PBS, circulation rate 120 ml/min, n ¼ 1). The data are presented as percentage of cell viability in the absence of Stx1.
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100 80 60 40
100
20
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(c)
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80 60 10min 30min 1h 2h 4h
0 0.0001
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0 0.001 0.01 0.1 1 10 100 1000 Stx1 concentration of circulated solution by dilution (b) (ng/ml)
Control
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0 0.001 0.01 0.1 1 10 100 1000 Stx1 concentration of circulated solution by dilution (ng/ml)
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(a)
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Cell viability (%)
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10
Stx1 concentration of circulated solution by dilution (ng/ml)
(d)
0.001
0.01
0.1
1
10
Stx1 concentration of circulated solution by dilution (ng/ml)
Fig. 5. The evaluation of the Stx1 adsorption ability by Dialyzers. (a) Control (no polymer attached) (condition; Stx1 concentration 4 mg/ml, solvent FCS, circulation rate 120 ml/min, mean7SE, n ¼ 3) (b) D-G2 (condition; Stx1 concentration 4 mg/ml, solvent FCS, circulation rate 120 ml/min, mean7SE, n ¼ 3). (c) Control (no polymer attached) (condition; Stx1 concentration 40 ng/ml, solvent FCS, circulation rate 120 ml/min, mean7SE, n ¼ 3). (d) D-G2 (condition; Stx1 concentration 40 ng/ml, solvent FCS, circulation rate 120 ml/min, mean7SE, n ¼ 3). The data are presented as the percentage of cell viability in the absence of Stx1.
the Stx1 concentration in the circulated solution was measured. The result of the control experiment showed that there was non-specific adsorption to FCS and/or dialyzer, but the circulated solution was still toxic (Fig. 5(c)). On the other hand, the result of D-G2 (Fig. 5(d)) showed the specific elimination. By circulating the solution for only 10 min, D-G2 mostly eliminated Stx1 even from the dilute Stx1 solution. This result showed that D-G2 had high specificity for Stx1 adsorption, and the protein in FCS was hardly adsorbed to immobilize G2. Consequently, dialyzer D-G2 has high potency of adsorbing Stxs in blood. 3.4. Western blotting for ascertaining adsorption of Stx1 Adsorption of Stx1 to the dialyzer and its specificity for a glycoconjugate polymer was ascertained. After Stx1–1% BSA–PBS solutions were circulated through D-G2 (dialyzer; globotriose polymer) and D-L2 (dialyzer; lactose polymer) for 4 h, D-G2 and D-L2 was washed sufficiently with PBS. Adsorbed Stx1 was eluted with 4 M MgCl2 solution from D-G2 and D-L2. The eluted solution was adapted to Western blotting and the result is shown in Fig. 6. The film obviously indicated a specific adsorption of Stx1 on the dialyzer. Stx1-based A and B subunit bands appeared only at the D-G2 lane. Consequently, Stx1
was caught by a globotrioses of G2 instead of acrylamide main chain. 3.5. Inhibition of the biological activities of Stxs by polymers All of the polymers were examined for the inhibition of cytotoxicity of Stxs (Fig. 7(a) and (b)). G2 (globotriose polymer) markedly inhibited the cytotoxic activity of Stxs. However, G3 and G4 (globotriose copolymers), as well as L2 (lactose polymer) have only low activity. C1 (cellobiose polymer) had no inhibitory effect for Stxs at any concentration. This result corresponded with the evaluations of the Stx1 adsorption abilities of D-G3, D-G4 (dialyzers; globotriose copolymers), and D-L2 (dialyzer; lactose polymer). Consequently, the reason that G3 and G4 did not have the inhibitory activity can be the same as the reason why D-G3 and D-G4 did not have the adsorption ability. The reason for this is that the glycoconjugate polymer without acrylamide, G2, and with acrylamide, G3 and G4 (globotriose copolymers), may each have a different conformation in aqueous solution. Furthermore, in order to prepare a dialyzer which can eliminate Stxs efficiently, it is necessary that a glycoconjugate polymer immobilized to a dialyzer has a high biological activity like G2.
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3.6. Kd of Stxs for polymer G2 by using BIAcore system These polymers G2 (globotriose polymer) and L2 (lactose polymer) were immobilized on a BIAcore chip. The surface of a sensor chip coated with CM-dextran (CM5) is similar to that of the cellulose hollow fibers except for the hydrophilicity. The Kd values were determined by Scatchard plot analysis. Stx1 and Stx2 had very low Kd (16.476.5 and 13.571.2 nmol/L) for G2. Consequently, Stx1 had a Kd value, which was the expected result of the dialyzer evaluation of D-G2 (dialyzer; globotriose polymer), for G2. The Kd value of Stx2 for G2 is anticipated and D-G2 also has the high ability of Stx2 adsorption. 4. Conclusion
Fig. 6. The evidence of the Stx1 adsorption for the dialyzers using Western blotting. (a) Standard. (b) The eluted solution from D-L2. (c) The eluted solution from D-G2. Stx1
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Cell viability (%)
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G2 G3 G4 L2 C1
We developed a Shiga-toxin elimination dialyzer which has immobilized glycoconjugate polymer containing globotriose. D-G2 (dialyzer; globotriose polymer) efficiently adsorbed Stx1 from both dilute and concentrated Stx1 solutions. Moreover, the dialyzer eliminated Stx1 from FCS solution, which has a high protein concentration. The inhibitory effects of the polymers on the cytotoxicity of Stx1 and Stx2 were also determined. G2 (globotriose polymer) showed high inhibitory effect on both Stx1 and Stx2. Kd values of G2, which were measured by BIAcore system, were in the nM range for Stx1 and Stx2. G2 showed appreciably favorable results for each of the evaluations. Significantly, D-G2 showed potential to eliminate Stx1 and Stx2 from blood.
60
References 40 20 0 0.000001 0.00001 0.0001
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Cell viability (%)
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10
100
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10
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0.001 0.01 0.1 Sugar content (µM)
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0.001 0.01 0.1 Sugar content (µΜ)
Fig. 7. Cytotoxicity assay. (a) using Stx1 (mean7SE, n ¼ 3). (b) using Stx2 (mean7SE, n ¼ 3). The data are presented as percentage of cell viability in the absence of Stx1.
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