Bioconjugate Chem. 1995, 6,361-366
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Galactose-Containing Amphiphiles Prepared with a Lipophilic Radical Initiator: Association Processes between Liposomes Triggered by Enzymatic Reaction? Kohji Ohno, Katsuko Sohda, Ayako Kosaka, a n d Hiromi Kitano" Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930, Japan. Received December 14, 1994@
A galactose-containing monomer (2-(methacryloyloxy)ethyl @-D-galactopyranoside,MEGal) was polymerized by using a lipophilic radical initiator. The amphiphile obtained formed a liposome by mixing with bis(truns,truns-2,4-dioctadecadienoyl)phosphatidylcholine(DDPC), and the liposome obtained was physically stabilized by the polymerization of DDPC by UV irradiation. The enzymatic treatment of the galactose-containing liposomes with galactose oxidase resulted in the formation of aldehyde groups on the liposome surface. By the subsequent mixing of the liposome suspension with the amino group-containing liposome suspension, a rapid increase in turbidity was observed due to the formation of Schiff bases between the aldehyde groups and the amino groups a t the interface of the liposomes. The rate of turbidity change strongly depended on the degree of polymerization of MEGal, the surface densities of galactose and amino groups on the liposome, the distance from the liposome surface to amino end groups, and the flexibility and deformability of the liposomes.
INTRODUCTION
EXPERIMENTAL PROCEDURES
Mutual recognition of cells in vivo is a n essential factor for the organization of tissues and organs and the immunological protection system ( I 1. In recent years, some potential techniques have been developed using the specific binding of ligand to receptor at the lipid-lipid interface (so-called cross-linked liposomes) (2-5). We have been studying the mutual recognition between liposomes whose surfaces are modified with complementary compounds (enzyme-enzyme inhibitor, biotinavidin, for example) (4, 5). Previously, Novogrodsky reported that lymphocyte cytotoxicity could be induced by treatment with neuraminidase and galactose oxidase or with the periodate of either the effector cells or the target cells, which initiated cross-linkage between the effector and target cells via a Schiff base (6). In this study, as a model system of this phenomenon, we examined the recognition of amino group-containing liposomes by galactose-containing liposomes that had been treated with galactose oxidase beforehand. Previously we prepared liposome-forming amphiphiles having pH or temperature responsiveness by the polymerization of acrylic acid or N-isopropylacrylamide by using a lipophilic radical initiator (7) or lipophilic chain transfer reagent (81, respectively. Using the lipophilic radical initiator (DODA-5011(Figure la)) and a galactosecontaining vinyl monomer (2-(methacryloyloxy)ethyl @-Dgalactopyranoside, MEGal (Figure lb)), we prepared galactose-containing amphiphiles with various degrees of polymerization (Figure IC). A lipid carrying only one galactose residue (Figure Id) was also prepared by the lactone method for comparison (9). By incorporating the galactose-containing amphiphile in liposomes, we could study the association processes of liposomes mediated by the Schiff bases, which were triggered by the enzymatic reaction.
Materials. A lipophilic radical initiator (DODA-501) was synthesized from Nfl-dioctadecylamine (DODA, Fluka, Switzerland) and 4,4'-azobis(cyanovaleric acid) (V501, Wako Pure Chemicals, Osaka, Japan) as described previously (7). A galactose-containing monomer, 2-(methacryloy1oxy)ethyl @-D-galactopyranoside (MEGal), was prepared by trans-glycosylation between o-nitrophenyl B-D-galactopyranoside and 2-hydroxyethyl methacrylate (HEMA) catalyzed by @-galactosidase(from Escherichia coli,340 unitdmg, Sigma, St. Louis, MO) in a phosphate buffer (1/15 M, pH 6.4) (IO)in the presence of hydroquinone. Anal. Calcd for C1~HzoOg1/2HzO:C, 47.84; H, 7.02; 0, 45.14. Found: C, 47.65; H, 6.88; 0, 45.47. lH NMR (DzO): 6 6.15 (s, lH, CHz=C 8.5 we could observe no change in turbidity (no aggregation of liposomes in other words), which shows the large pH sensitivity of association processes between the liposomes. By using amino group-containing liposomes with various ratios of GGA and DMPC, we could examine the effect of the surface concentration of amino groups on the rate of turbidity change. As shown in Figure 6, the rate of turbidity change increased with the surface concentration below about 9 mol % of GGA in the liposome and then decreased significantly. The rate of the condensation reaction to produce the Schiff base is known to be the largest in the pH range 3-5 (20).As a possibility, the pH in the vicinity of the liposome surface with a high content of GGA might become higher than that in bulk solution, which makes it more difficult to form Schiff bases between two kinds of liposomes. Therefore, the surface concentration of amino groups strongly affects the rate of turbidity change. This result is consistent with the pH sensitivity of association processes described earlier. In conclusion, primary hydroxyl groups of galactose residues bound to the polymer chains existing on the liposome surface could be oxidized by galactose oxidase and converted into aldehyde groups. The Schiff bases formed between aldehyde groups and amino groups on the other liposome surfaces resulted in aggregation of the
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Content of DODA-PMEGal (mol%) Figure 4. Effect of mixing ratio of DDPC and DODA-PMEGal (DP = 15) in the liposome on the rate of turbidity change after injection of the amino group-containing liposome suspension into the galactose-containing liposome that had been incubated with galactose oxidase for 30 min at 37 "C beforehand: (0) DMPC/ GGA liposome; ( 0 )DMPCIGA liposome; (0)DPPC/GGA liposome; (m) DPPCIGA liposome. The content of amino groupcontaining lipid was 10 mol %. The concentrations of both liposomes were 100 pg of lipid/mL. The concentration of enzyme was 0.1 unit/mL (HEPES (10 mM, pH 6.0) buffer; wavelength = 350 nm).
then increased steeply with the concentration, which suggests the presence of a threshold value in the recognition of aldehyde groups by amino group-containing liposomes. This result is in accordance with the steep increase in the turbidity 15 min after the onset of the reaction observed in Figure 2a. To realize the stable binding between two kinds of liposomes, the multiple formation of Schiff bases between many aldehyde groups and amino groups might be necessary, thus bringing about a threshold phenomenon. To examine the effect of the surface concentration of galactose residues on the rate of turbidity change, liposomes with various molar ratios of DODA-PMEGal (DP = 15) and DDPC were prepared. Figure 4 shows that the rate of turbidity change increased with the content of DODA-PMEGal (DP = 151, in contrast to the results of Figure 3. This is probably because the catalytic behavior of the enzyme a t interfaces is more complicated compared to that with the free substrate in solution. Further findings about this will be reported in the near future. We also investigated the role of lipid composition on membrane-membrane dissociation reactions: The suspension of the galactose-containing liposome with various molar ratios of DODA-PMEGal (DP = 8.4) and DDPC was incubated with galactose oxidase for 60 min at 37 "C. After the liposome suspension was mixed with the amino group-containing liposome suspension, the turbidity increased and leveled off. Subsequently, 0.05 N NaOH was injected into the turbid mixture. The rates of decrease in turbidity had no obvious differences in the range of concentration ratios in which these experiments were carried out (DODA-MEGal, 1.7-6.6 mol %; GGA, 10 mol %I (data not shown). The number of galactose residues on the liposome surface even a t the lowest molar ratio (DODA-PMEGal (DP = 8.41,1.7 mol %) was about 1.4 times as many as that of the amino groups on the liposome surface. Since the growth of the multiple formation of Schiff bases would be restricted due to the steric hindrance, the number of Schiff bases formed until
Ohno et al.
366 Bioconjugate Chem., Vol. 6,No. 4, 1995
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appreciated for the kind donation of DDPC. This work was supported by a Grant-in-Aid (06453153)from the Ministry of Education, Science and Culture.
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LITERATURE CITED
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PH Figure 5. Effect of pH in the solution on the rate of turbidity change after injection of the amino group-containing liposome suspension with various pH values into the galactose-containing liposome that had been incubated with galactose oxidase for 30 min at 37 "C beforehand. The contents of DODA-PMEGal (DP = 8.4) and GGA were 4.5 and 10 mol %, respectively. The concentrations of both liposomes were 100 pg of lipid/mL. The concentration of enzyme was 0.1 unit/mL. The galactosecontaining liposome was incubated with the enzyme in HEPES (10 mM, pH 6.0) buffer (wavelength = 350 nm).
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Content of GGA (mol%) Figure 6. Effect of the mixing ratio of DMPC and GGA in the liposome on the rate of turbidity change after injection of the amino group-containing liposome into the galactose-containing liposome that had been incubated with galactose oxidase for 30 min at 37 "C beforehand. The content of DODA-PMEGal (DP = 15)was 4.5 mol %. The concentrations of both liposomes were 100 pg of lipid/mL. The concentration of enzyme was 0.1 unit/ mL (HEPES (10 mM, pH 6.0) buffer; wavelength = 350 nm).
liposomes. The rate of turbidity change depended on the densities of galactose and amino groups on the liposome sudaces, the distance from the liposome surface to the galactose residue and amino end groups, and the membrane flexibility and deformability. The results obtained also suggest the importance of morphology of the cell surface in cell-cell recognition phenomena such as lymphocyte cytotoxicity. ACKNOWLEDGMENT
We are grateful to Wako Pure Chemicals for their kind donation of V-501. The Japan Fat and Oil Co. is greatly
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