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Bioconjugate Chem. 1998, 9, 50−53
Capture of HIV-1 gp120 and Virions by Lectin-Immobilized Polystyrene Nanospheres† Mitsuru Akashi,*,‡ Takashi Niikawa,‡ Takeshi Serizawa,‡ Tadao Hayakawa,§ and Masanori Baba*,§ Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890, Japan, and Division of Human Retoroviruses, Center for Chronic Viral Diseases, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890, Japan. Received March 28, 1997; Revised Manuscript Received October 7, 1997X
A lectin such as concanavalin A (Con A) was immobilized on the surfaces of poly(methacrylic acid) branches covered with polystyrene nanospheres with a diameter of 360 nm, which were obtained by the free radical copolymerization of styrene with the poly(tert-butylmethacrylate) macromonomer, followed by hydrolysis. Using Con A-immobilized (0.3 µg/cm2) nanospheres, the interaction of the nanospheres with HIV-1 was determined by the reduction of the gp120 level and the viral infectivity of the HIV-1 suspensions after a 60 min incubation at room temperatures. Con A-immobilized nanospheres achieved a 95 and a 77% reduction of the gp120 level and the infectivity at a concentration of 0.5 mg/mL, respectively, indicating the effective capture of the gp120 and the virions.
INTRODUCTION
Human immunodeficiency virus-1 (HIV-1) is a spherical virus with a diameter of 100 nm and possesses an envelope with glycoprotein gp120 and gp41 (1). Gp120 is a mannose-rich glycoprotein, so it strongly interacts with some kinds of lectins (2, 3). In particular, concanavalin A (Con A) is reported to have a high affinity for gp120. Thus, there is a strong possibility that gp120 and HIV-1 virions will be effectively captured by lectins, if they are ideally immobilized to certain materials. Actually, purification of the HIV envelope glycoprotein by means of Con A-agarose affinity chromatography was reported (4). Polymeric materials such as gels, films, beads, and particles are frequently used for the immobilization of biomolecules such as antibodies and enzymes in order to realize immuno assays (5, 6) and catalytic systems (7). Among these, polystyrene beads and particles are quite useful because of the ease of their preparation. Since there is no functional group on their surfaces, the immobilization of biomolecules by means of covalent bonds is inadequate. In addition, as the surfaces of the polystyrene beads and the particles are highly hydrophobic, nonspecific adsorption of other biomolecules seems to be unavoidable. To circumvent these problems, we recently developed technology for hydrophilic polymer chains with functional group-coated polystyrene nanospheres (8, 9), which is dispersed well in water and very useful for biomolecule immobilization. Consequently, these nanospheres are quite useful as immunolatex, when antibodies, for example, are introduced into surface† This paper is part XIV in the series of the study on Graft Copolymers Having Hydrophobic Backbone and Hydrophilic Branches. Part XIII is as follows: Serizawa, T., and Akashi, M. (1997) Chem. Lett., 809. * To whom correspondence should be addressed. ‡ Department of Applied Chemistry and Chemical Engineering. § Division of Human Retroviruses. X Abstract published in Advance ACS Abstracts, December 15, 1997.
modified nanospheres. Moreover, the peptide drug calcitonin shows a pharmaceutical activity in oral administration when it has been physically immobilized onto polystyrene nanospheres coated with water-soluble polymer chains (10). In this paper, we will discuss the immobilization of lectins onto surface-modified polystyrene nanospheres and their capture of HIV-1 particles through affinity interaction between gp120 mannose and the immobilized lectins. RESULTS AND DISCUSSION
First, poly(methacrylic acid)-covered polystyrene nanospheres were prepared by the copolymerization of styrene with the poly(tert-butyl methacrylate) macromonomer and the following acid hydrolysis, and analyzed according to the method described previously (8). Because methacrylic acid oligomers contain many active carboxyl groups that are inadequate for macromonomer synthesis, we prepared the poly(tert-butyl methacrylate) macromonomer by the reaction between poly(tert-butyl methacrylate) oligomers and p-vinylbenzyl chloride. The resulting nanospheres (mean diameter of 360 nm) were dispersed well in water and were completely collected by centrifugation or freeze-drying after dialysis. Electron spectroscopy for chemical analysis (ESCA) of the surface suggested that poly(methacrylic acid) chains were favorably located on the surfaces of the polystyrene particles. Scheme 1 illustrates the immobilization of Con A onto the surface of the polystyrene nanospheres. Lectins were immobilized on the surface of polystyrene particles by means of their amino group. Using water-soluble carbodiimide (WSC), the condensation between the carboxyl group of poly(methacrylic acid) on the polystyrene surface and the amino group of Con A gave Con A-immobilized polystyrene nanospheres in a 0.01 M HEPES buffer solution (pH 8.0) at 4 °C for 24 h. After reaction, the centrifuged nanospheres were repeatedly washed in order to correct the Con A-immobilized nanospheres. The surfaces of the obtained nanospheres were fully covered with Con A. To investigate how Con A-immobilized polystyrene nanospheres can capture HIV-1, they were added to an
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Capture of HIV-1 gp120 and Virions
Bioconjugate Chem., Vol. 9, No. 1, 1998 51
Scheme 1. Immobilization of Lectins onto the Surface of Polystyrene Nanospheres
Scheme 2. Capture of HIV-1 Virions by LectinImmobilized Polystyrene Nanospheres
Figure 1. Effect of Con A-immobilized nanospheres on the (A) gp120 and (B) viral infectivity of HIV-1. The medium containing HIV-1 was mixed with an equal volume of (1) control medium, (2) control nanospheres (final concentration of 0.5 mg/mL), or (3) Con A-immobilized nanospheres (final concentration of 0.5 mg/mL). After a 60 min incubation at room temperature, the mixtures were centrifuged, and the supernatants were examined for their gp120 antigen level and viral infectivity. Data represent mean values in triplicate experiments. Table 1. HIV-1 Capturing Using Various Lectin-Immobilized Nanospheresa
HIV-1 medium, as shown in Scheme 2. The suspension (0.5 mL) of the Con A-immobilized nanospheres (final concentration of 0.5 mg/mL) was added to an equal volume of the medium that contained HIV-1 (IIIB strain), and the mixed suspension was incubated at room temperature for 60 min. It was assumed that the Con A-immobilized nanospheres would be aggregated, if the HIV-1 virions or the gp120 could interact with the nanospheres. In fact, the Con A-immobilized nanospheres but not the control nanospheres apparently aggregated. Consequently, the Con A-immobilized nanospheres were easily precipitated by centrifugation (Scheme 2). The amount of captured HIV-1 was determined by the residual gp120 antigen level and the viral infectivity in the supernatants after centrifugation at 8200g for 10 min. The gp120 level was measured by using an HIV-1 gp120 antigen capture ELISA kit (Advanced Biotechnologies, Columbia, MD). Viral infectivity was assessed by microscopic observation for HIV-1-induced cytopathicity in MT-4 cells and expressed as a 50% cell culture infectious dose (CCID50). In these assay systems, Con A-immobilized nanospheres achieved a 95 and a 77% reduction of the gp120 level and the viral infectivity at a concentration of 0.5 mg/mL, respectively (Figure 1). In contrast, there was only a slight reduction of the gp120 level and the viral infectivity was identified for the nanospheres that were not immobilized with Con A (Figure 1). Since a small amount of free Con A was contaminated with the suspension of the Con A-immobilized nanospheres, we also examined the effects of the free Con A on the HIV-1 capture assays. The contamination-free Con A (0.79 µg/ mL) did not significantly affect the gp120 level or the viral infectivity (data not shown). In order to know the capture activity using other lectins, the mannose-sepecific lectins such as lens culinaris agglutinin (LCA), galanthus nivalis lectin (GNL), and hippeastrum hybrid lectin (HHL) and also the galactose-specific recinus communis agglutinin (RCA120) were immobilized onto the nanospheres by the same procedure. The amounts of lectins immobilized onto the nanospheres and their capture
b
lectin
immobilized amount (pmol/cm2)
HIV-1 capturingb (%)
Con A LCA GNL NHL RCA120
3.18 2.44 2.08 1.10 7.30
95 89 82 83 39
a The concentration of their nanospheres was 0.5 mg/mL. Estimated by the ELISA technique.
Figure 2. Dose-dependent HIV-1 capture by Con A-immobilized nanospheres. The medium containing HIV-1 was mixed with an equal volume of (1) control medium or Con A-immobilized nanospheres at a final concentration of (2) 0.02, (3) 0.1, and (4) 0.5 mg/mL. After a 60 min incubation at room temperature, the mixtures were centrifuged, and the supernatants were examined for their gp120 antigen level and viral infectivity. Data represent mean values in triplicate experiments.
activities are listed in Table 1. The LCA-, GNL-, and HHL-immobilized nanospheres still performed effective capture activity, even though smaller amounts of their lectins compared to Con A were immobilized. The steric hindrance or direction of binding pockets as well binding specificity of each lectin will be significant for their activity. Also, the RCA120-immobilized nanospheres showed little effect on the HIV-1 capturing as expected from its specificity. Figure 2 shows dose-dependent HIV-1 capture activity of Con A-immobilized nanospheres. Even at a concentration of 0.1 mg/mL, the Con A-immobilized nanospheres reduced the gp120 level by 50%. To determine the specificity of the Con A-immobilized nanospheres, we conducted an inhibition assay
52 Bioconjugate Chem., Vol. 9, No. 1, 1998
Figure 3. Mannose inhibits HIV-1 capture by Con A-immobilized nanospheres. The medium containing HIV-1 was mixed with an equal volume of (1) control medium or Con A-immobilized nanospheres (final concentration of 0.5 mg/mL) containing mannose at a final concentration of (2) 0, (3) 10, (4) 100, and (5) 250 mg/mL. After a 60 min incubation at room temperature, the mixtures were centrifuged, and the supernatants were examined for their gp120 antigen level and viral infectivity. Data represent mean values in triplicate experiments.
for HIV-1 capture by the addition of mannose. When an excess amount of mannose was added to the Con Aimmobilized nanospheres, their capture activity was weakened in a dose-dependent fashion (Figure 3), indicating that the Con A-immobilized nanospheres primarily recognize the mannose molecules in gp120. Furthermore, capture activity was not hampered by the addition of galactose (data not shown). In this study, we have shown that HIV-1 particles and the gp120 antigen can be effectively captured by lectinimmobilized nanospheres. Thus, the lectin-immobilized nanospheres may be potential chemotherapeutic or prophylactic agents in HIV-1 infection. Apparently, several issues, including efficacy and toxicity in vivo, still need to be elucidated. The selection of immobilized lectins and the diameter of nanospheres are important determinants for their capture activity and selectivity. In fact, nanospheres that immobilized mannose-specific lectins other than Con A were found to be equally effective with a smaller amount of immobilization (data not shown). Further research is now in progress that will attempt to obtain more effective and selective nanospheres for the capture of HIV-1. EXPERIMENTAL PROCEDURES
Materials. Styrene (Wako Pure Chemical Ind., Tokyo, Japan) and tert-butyl methacrylate (Wako Pure Chemical Ind.) were used after distillation under reduced pressure. 2-Mercaptoethanol (2-MEtOH) (Wako Pure Chemical Ind.), sodium hydride (NaH) (Nacalai Tesque, Kyoto, Japan), and tetrabutylphosphonium bromide (TBPB) (Wako Pure Chemical Ind.) were used without further purification. 2,2′-Azobis(isobutyronitrile) (AIBN) was recrystallized from methanol and dried under vacuum. Tetrahydrofuran (THF), N,N-dimethylformamide (DMF), ethanol, water, and other solvents were distilled by the ordinary ways. p-Chloromethylstyrene (CMSt) was a gift from Nippon Oil and Fats Co. (Tokyo, Japan) and used without further purification. Preparation of tert-Butyl Methacrylate Oligomers. tert-Butyl methacrylate oligomers that were terminated with a hydroxyl group were prepared by the free radical polymerization of tert-butyl methacrylate with AIBN as an initiator in the presence of 2-mercaptoethanol as a chain transfer reagent (8). For instance, into a 200 mL three-necked round-bottomed flask equipped with a mechanical stirrer, a condenser, and a nitrogen
Akashi et al.
inlet tube were placed 176 mmol of tert-butyl methacrylate, 0.176 mmol of AIBN, and 2.18 mmol of 2-mercaptoethanol in 60 mL of THF. After dry nitrogen gas was bubbled into the reaction media at 0 °C for 30 min, it was kept at 60 °C for a further 6 h. The reaction mixture was poured into H2O/methanol (1/1, v/v). The precipitated polymer was filtered off and dried under reduced pressure. The polymer was resolved again in 2-propanol, and then the solution was poured into H2O/methanol (1/ 1, v/v). The purification procedure was repeated several times. The yield was 74%. The Mn and Mw/Mn were 3870 and 2.07, respectively. Preparation of Poly(tert-butyl methacrylate) Macromonomers. The macromonomer was prepared by combination of CMSt with a hydroxyl group of the oligomer under an alkaline condition (8). The 0.78 mmol of the tert-butyl methacrylate oligomer was stirred with a 10-fold molar excess of NaH in 100 mL of DMF at 30 °C for 30 min. After that, the 10-fold molar excess (7.8 mmol) of CMSt was added into the flask and the mixture stirred for another 48 h in the presence of TBPB as a phase transfer catalyst. The reaction mixture was poured into H2O/methanol (1/1, v/v). The precipitated polymer was filtered off and dried under reduced pressure. The polymer was resolved again in 2-propanol, and then the solution was poured into H2O/methanol (1/1, v/v). The purification procedure was repeated several times. The yield was 84%. The Mn and Mw/Mn were 4100 and 1.97, respectively. Preparation of Poly(tert-butyl methacrylate)Covered Polystyrene Nanosphere. The poly(tertbutyl methacrylate)-covered polystyrene nanosphere was prepared by the free radical polymerization of the corresponding macromonomer and styrene in a polar solvent (8). The 0.115 mmol of the poly(tert-butyl methacrylate) macromonomer and the 40-fold molar excess (4.6 mmol) of styrene were weighted into a glass tube with 0.047 mmol of AIBN and 5 mL of ethanol, and then the mixture was degassed by freeze-thaw cycles on a vacuum apparatus, sealed off, and kept at 60 °C for 24 h. The reaction medium was dialyzed in methanol by using a cellulose dialyzer for 5-6 days. The nanosphere was obtained by lyophilization. The yield was 41%. The particle size and its standard deviation of the size were 199 and 34 nm, respectively. Preparetion of Poly(methacrylic acid)-Covered Polystyrene Nanosphere. The poly(methacrylic acid)covered polystyrene nansopshere was prepared by hydrolysis of the poly(tert-butyl methacrylate) that was introduced on the poly(tert-butyl methacrylate)-covered polystyrene nanosphere under an acidic condition (8). An adequate amount of the poly(tert-butyl methacrylate)covered polystyrene nanosphere was dispersed in a 12 N HCl water/ethanol (1/5, v/v) solution. The dispersion was heated at 70 °C for 24 h. The reaction mixture was dialyzed in water for several days. The particle size was 360 nm, which was larger than that of the poly(tert-butyl methacrylate)-covered polystyrene nanosphere. It seemed that electrostatic repulsion of anionic poly(methacrylic acid) on the nanosphere enlarged its size. The standard deviation was 120 nm, which means its size distribution became slightly larger with the acid hydrolysis of the poly(tert-butyl methacrylate). Immobilization of Con A onto Poly(methacrylic acid)-Covered Polystyrene Nanosphere. A carboxyl group of polystyrene nanospheres (2.5 mg) was first activated by WSC (1.0 mg/mL in 0.05 M KH2PO4) for 30 min. The nanospheres that were obtained by centrifugation were mixed with 1.0 mg of lectin in 1.0 mL of 0.01
Capture of HIV-1 gp120 and Virions
M HEPES buffer solution (pH 8.0), and the mixture was kept at 4 °C for 24 h. After reaction, the centrifuged nanospheres were repeatedly washed. The amount of immobilized lectin was evaluated by means of the ninhydrin method. The surfaces of the obtained nanospheres were covered with 0.3 µg of Con A/cm2. Characterization Method. The number-average molecular weights (Mn) of the oligomer and macromomnomer were determined by GPC (Shimadzu LC-6A system with a Shodex AC-800P column), calibrated with polystyrene standards. The terminal vinylbenzyl group of the macromonomer was confirmed by 1H-NMR spectra obtained with a JEOL FX-400 (400 MHz) instrument. Particle-size measurements were achieved by direct observation with a scanning electron microscope (SEM) (Hitachi H-7010A) and a submicron particle analyzer (Coulter model N4SD). Surface analysis by ESCA was carried out a Shimadzu ESCA 1000 apparatus. Viruses. The IIIB strain of HIV-1 was used in the experiments. The virus was propagated in MOLT-4 cells. The infected cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics (culture medium), and their culture supernatants were centrifuged at 3000 rpm for 10 min, filtered, and stored at -80 °C until they were used. The infectivity of viral stocks was determined in MT-4 cells, and their gp120 antigen level was measured by a gp120 antigen-capture ELISA kit (Advanced Biotechnologies). ACKNOWLEDGMENT
This work was financially supported in part by a Grant-in-Aid for Scientific Research in Priority Areas of New Polymers and Their Nano-Organized Systems (277/ 08246243) and a Grant-in-Aid for Scientific Research (08458291) from the Ministry of Education, Science, Sports and Culture, Japan. LITERATURE CITED (1) Evans, L. A., and Levy, J. A. (1993) The heterogeneity and pathogenicity of HIV. In HIV Molecular Organization, Pa-
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BC970045Y
