Bioconjugafe Chem. 1995, 6, 131-1 34
131
Galactose-Containing Amphiphiles Prepared with a Lipophilic Radical Initiator? Hiromi Kitano,* Katsuko Sohda, a n d Ayako Kosaka Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930,Japan. Received June 27, 1994@
Novel amphiphiles which contain galactose residues (degree of polymerization (DP) = 6.2,10, and 15) were prepared by telomerization of 2-[(methacryloyloxy)ethyll-/3-~-galactopyanoside using a lipophilic radical initiator. The galactose-carrying amphiphile incorporated in a liposome was recognized by a lectin from Ricinus communis ( R C A I ~ ~ which ) , was proven by the increase in turbidity of the liposome suspension after mixing with the lectin. The recognition was largely affected by the degree of polymerization and the surface density of the amphiphile. The amphiphile would be useful as a component of the drug delivery system to hepatocytes.
INTRODUCTION Hybrid materials, which are conjugates of a plural number of functional moieties, are very useful as intelligent materials in many research and application fields (1). Devices used in drug delivery systems, for example, have very often been included in a category of hybrid materials because various components (designed for targeting, encapsulation, slow release, reduction of immunogenicity, and pharmacological activity, etc.) have to be combined to prepare the systems which are practically useful (2). Previously we prepared liposome-forming amphiphiles having pH- or temperature-responsiveness by polymerization of acrylic acid or N-isopropylacrylamide by using a lipophilic radical initiator (3)or lipophilic chain transfer reagent (41,respectively, or by copolymerization of polymerizable lipid with N-methacryloyl-D,L-homocysteine thiolactone (5). These amphiphiles would be strong candidates to be used in drug delivery systems. Galactose residues are very important to be recognized by lectin-like proteins located on the surface of parenchymal hepatocytes (6). In this paper, therefore, we prepared amphiphilic compounds with several (6.2, 10, and 15) galactose residues in their hydrophilic head groups by telomerization of a galactose-containing vinyl monomer using the lipophilic radical initiator. We examined a recognition of the amphiphilic compounds by a lectin in a bilayer system. For comparison, a lipid carrying only one galactose residue was also prepared by the lactone method (7). The galactose-carrying lipophilic compounds examined here would be highly useful as devices for targeting to hepatocytes.
Chart 1. Chemical Structures of Monomer, Initiator, and Amphiphiles 0
OH (B) DODA-501 ClaH37\
P
FN FN 0 N-C-CH2-CH2-C-N=N-C-CHz-CH2-C-N
C18H37/
CH3
CH3
1 C18H37 \ Cl8H37
(C)DODA-PMEGal
dioctadecylamine (DODA, Fluka, Switzerland) and 44'azobis(cyanova1ericacid) (V-501, Wako Pure Chemicals, Osaka, Japan) as previously reported (3). N-DiglycylNJV-dioctadecylamide (GGA) was prepared by the reaction of N-benzyloxycarbonyl (2)-diglycine p-nitrophenyl EXPERIMENTAL PROCEDURES ester with dioctadecylamine in THF and subsequent Materials. 2-[(Methacryloyloxy)ethyll-/3-~-galactopy- deprotection of the Z group by the HBr-CH3COOH method (5). A lectin, Ricinus communis agglutinin ranoside (MEGa1,l Chart 1A) was prepared by the transglycosylation between p-nitrophenyl /3-D-galactopyranoAbbreviations: DLS, dynamic light scattering; DMF, diside and 2-hydroxyethyl methacrylate (HEMA)catalyzed methylformamide; DMPC, L-a-dimyristoylphosphatidylcholine; by /3-galactosidase (from E. coli, 340 d m g , Sigma, St. DODA, dioctadecylamine; DODA-L-Gal, dioctadecylamineLouis, MO) in a phosphate buffer (1/15 M, pH 6.4)(8)in galactose conjugate prepared by lactone method; DODAthe presence of hydroquinone. A lipophilic radical initiaPMEGal, dioctadecylamine-poly(2-[(methacryloyloxy)ethyll-~tor (DODA-501, Chart 1B) was prepared from NJVD-galactopyranoside)conjugate; DODA-501, azo radical initiator with dioctadecyl groups; DP, degree of polymerization; GGA, * To whom correspondence should be addressed. N-diglycyl-NJV-dioctadecylamide;HEMA, 2-hydroxyethyl meth+ Presented at the Regional Meeting of the Society of Polymer acrylate;MEGal, 2-[(methacryloyloxy)ethyll-~-~-galactopyranoside; MeOH, methanol; RCA or RCA120, Ricinus communis Science, Japan, Toyama University, October 1994. Abstract published in Advance ACS Abstracts, January 1, agglutinin; THF, tetrahydrofuran; V-501, 4,4'-azobis(cyanova1995. leric acid);Z, N-benzyloxycarbonyl. @
1043-1802/95/2906-0131$09.00/0 0 1995 American Chemical Society
132 Sioconjugate Chem., Vol. 6,No. 1, 1995
Kitano et al.
Chart 2. Chemical Structure of DODA-L-G~I Q
f
C-N-CH2-C-N-CH2-C-N
HOCHp
,Cl8H37
'
ClaH37
y-l
OH
(RCAIZO), and L-a-dimyristoylphosphatidylcholine (DMPC) were from Sigma. Other reagents were commercially available. A Milli-Q grade water was used for preparation of sample solutions.
Table 1. Preparation of DODA-PMEGal DODA-501 amphiphile (g) DODA-PMEGal-1 0.031 DODA-PMEGal-2 0.153 DODA-PMEGal-3 0.167
product MEGal solvent (mg) (g) (mL) [yieldlb DP" 0.112 2Oe 22 [191 6.2 0.692 20d 129 [ l l ] 15 0.191 30d 23 L2.51 10
a Degree of polymerization. * In regard to DODA-501(%). THF. DMF.
thin lipid membrane formed was dispersed into a phosphate buffer (pH 7.2) using a vortex mixer. The dispersion was further sonicated by a n ultrasonifier (Astrason W-385, Heat Systems-Ultrasonics, Inc., NY)for 3 min while NZ gas was passed through the suspension. The liposome suspension was finally passed through a syringe filter (Millipore Millex-GV, pore size; 0.22 pm).
Preparation of Galactose-ContainingAmphiphile by Polymerization. DODA-501 (31 mg) and MEGal (112 mg) were dissolved in THF (20 mL) in a test tube. After NZgas was passed through the solution for several minutes, the reaction mixture was tightly sealed and incubated for 24 h a t 70 "C. After evaporation of the Release of Eosin Y Incorporated in Liposomes. solvent, the sugar-containing amphiphile was purified by To confirm the presence of liposomal structure in the washing with n-hexane (to remove unreacted initiator) dispersion mixture of DODA-PMEGal and DMPC, Eosin and subsequently with cold water (to remove polymers Y was dissolved in the phosphate buffer to disperse the without lipophilic end group). The amphiphile dispersed lipids. After separation of liposomes from free Eosin Y in water by a vortex mixer was further purified by by gel permeation chromatography (Sephacryl S-1000,15 passing through a GPC column (2 x 20 cm, Sephacryl (i.d.1 x 200 mm), the liposome suspension was sonicated S-200, mobile phase; water) and lyophilized (22 mg, for 3 min a t 60 "C to disrupt the liposomes. The DODA-PMEGal, Chart 1C) (IR, OH stretching of galacfluorescence intensity of the suspension a t 555 nm tose 3350-3500 cm-l, C s N stretching 2050 cm-', C=O (excitation, 305 nm) before and after the sonication was stretching of ester bond 1720 cm-', C-0 stretching of compared. Eosin Y is well known to show a significant tertiary amide 1630 cm-', CH2 vas 2950 cm-l, CHz v, 2870 fluorescence by dilution (3,5)due to a reduction of selfcm-l). The degree of polymerization (DP) of the amquenching phenomenon. phiphile was determined as 6.2 by elemental analyses. Dynamic Light Scattering Method. Hydrodynamic Anal. Calcd for C 4 ~ H ~ ~ N z O ( C 1 ~ H z oC, O ~57.20; ) ~ , ~ : H, diameter of liposomes composed of various molar ratios 8.50; N, 1.14. Found: C, 57.23; H, 8.68; N, 1.15. By of galactose-lipid and DMPC was determined by the using a similar method, the amphiphiles with DP = 10 dynamic light scattering (DLS) method (DLS-7000, Otand 15 were prepared. DODA-PMEGal (DP = 10). suka Electronics, Hirakata, Japan; light source, He-Ne Anal. Calcd for C42HszNzO(ClzHzo08)1o: C, 54.75; H, 8.00; laser 632.8 nm). N, 0.79. Found: C, 54.53; H, 8.25; N, 0.77. DODAPMEGal (DP = 15). Anal. Calcd for C ~ Z H ~ Z N Z O - Turbidity Measurements. The lectin-induced agglutination of liposomes was followed by the increase in (C1zHzoOs)ls: C, 53.16; H, 7.68; N, 0.56. Found: C, 53.13; decadic absorbance at 450 nm by using a UV-vis H, 7.74; N, 0.56. spectrophotometer (Ubest-35, Japan Spectroscopic Co., Preparation of Galactose Lipid by the Lactone Tokyo, Japan). The observation cell was thermostated Method. Lactose monohydrate (12 g) was oxidized by by a Peltier device. The uncertainties of the rate of iodine (17.1 g) in the presence of KOH (16 g) (solvent; turbidity change were within 30%. 665 mL of MeOH and 25 mL of HzO) a t 40 "C. The precipitated solid was purified by recrystallization from RESULTS AND DISCUSSION MeOH-Hz0 (125). The crystal was dissolved in HzO, and the solution was passed through a n ion-exchange A. Galactose-CarryingAmphiphiles. The yields column (2.5 x 30 cm, Amberlite IR-120 B, H+ form) of sugar-containing lipids were in general very low several times. The aqueous solution of the product was because of the difficulty to purify the amphiphile commixed with EtOH, and the solvent was evaporated a t 70 pounds (Table 1). The increase in fluorescence intensity "C to form a lactose-lactone (5.45 g, 48% yield) (IR, C-0 of the dispersion of DODA-PMEGal and DMPC due to stretching of lactone ring 1730 cm-l). N-Diglycyl-NJVa release of Eosin Y from inner water pool to the bulk dioctadecylamide (GGA, 0.406 g) was coupled with the solution definitely proved the liposomal structure. Using lactone (0.651 g) in DMF-CHC13 (3:1, 40 mL) a t 60 "C the DLS method, diameter of the liposomes composed of for 6 h. The galactose-carrying lipid was purified by DODA-PMEGal and DMPC was estimated to be 1400 precipitation in hexane-EtOH (2:1).The precipitate was A on average. further dissolved in CHC13 and passed through a glass B. Recognition of Galactose Residues on Lipofilter. The filtrate was finally evaporated to give a somes by Lectin. Turbidity of galactose-carrying lipslightly yellow powder (DODA-L-G~(Chart 21, 21 mg, osomes a t 450 nm was rapidly increased by the addition 3.4%yield). I R OH stretching of sugar 3300-3400 cm-', of lectin, probably due to a recognition of galactose C-0 stretching of tertiary amide 1680 cm-l, C=O residues on the liposome surface by the lectin and stretching of secondary amide 1640 cm-l, NH deformasubsequent aggregation of liposomes mediated by the tion of secondary amide 1520-1540 cm-l, CHz vas 2920 lectin molecules. cm-l, CHZv, 2850 cm-l. Anal. Calcd for C5zH101013N3: By the addition of lactose to the liposome suspension, C, 63.97; H, 10.43; N, 4.30. Found: C, 63.76; H, 10.38; this aggregation of liposomes was largely inhibited N, 4.55. (Figure 1). Furthermore, the addition of lactose or Preparation of Liposome. The galactose-containing galactose to the suspension of galactose-liposome-RCA lipid and DMPC with various molar ratios were dissolved aggregates induced the reduction of turbidity: The addition of 0.45 and 18.2 mM of galactose reduced in CHC13 (5 mL), and the solvent was evaporated. The
Bioconjugate Chem., Vol. 6,No. 1, 1995 133
Galactose-ContainingAmphiphiles
0
0.1
0.2
0.3
[ Lactose ] ( mM )
Figure 1. Inhibitory effects of lactose on the rate of turbidity change in the agglutination of galactose-containing liposomes by RCA: ( 0 )DODA-L-G~;( 0 )DODA-PMEGal (DP = 15).
mol% glycolipid Figure 2. Effect of galactose lipid content in the liposome on the rate of turbidity change after the addition of RCA into the liposome suspension: ( 0 )DODA-L-Gal; (A)DODA-PMEGal (DP = 6.2); ( x ) DODA-PMEGal (DP = 10);(0)DODA-PMEGal (DP = 15). [RCA] = [lipid] = 0.05 mg/mL.
turbidity of the suspension of aggregated liposomes by 5% and 33% in 1 min, respectively, and that of lactose reduced the turbidity by 29%and Mi%,respectively. The reduction effect of lactose was much larger than that of galactose, which was in agreement with the tendency in the aggregation of lactosylceramide-containingliposomes by RCA (9). These results strongly support that the turbidity change is due to the specific recognition of galactose residues on the liposomes by RCA. The turbidity change of galactose-liposomes by the addition of RCA was dependent on the molar ratio of the galactose lipid and DMPC. Figure 2 shows that the increase in percents of galactose lipid induced the very gradual increase in the rate of turbidity change in the cases of DODA-L-Gal and DODA-PMEGal (DP = 6.2), whereas there was a relatively steeper increase in the case of DODA-PMEGal (DP = 15). The presence of threshold value in mol % of lactosylceramide in liposomes a t the lectin-induced aggregation was previously reported (9). The tendency observed in the DODA-L-G~liposome and DODA-PMEGal (DP = 6.2) liposome systems (very small increase in the rate of turbidity change a t low glycolipid contents) is not inconsistent with that previously reported. In the case of the DODA-PMEGal (DP = 15) liposome system, however, the increase was relatively much steeper because many galactose residues are present in a polar head region of one lipid molecule, which makes it unnecessary for the lectin to bind to a plural number of sugar lipids on each liposome t o realize stable aggregation and, consequently, strengthens the
[ RCA I ( mg/ml )
Figure 3. Effect of RCA concentration on the rate of turbidity change after the addition of RCA into the liposome suspension: (0)DODA-L-Gal; (A)DODA-PMEGal (DP = 6.2); ( x ) DODADODA-PMEGal (DP = 15). Galactose PMEGal (DP = 10);(0) lipid, 9.65 mol %. [Lipid] = 0.05 mg/mL.
agglutinability of the lectin a t a low content of galactoselipid. Furthermore, the turbidity change by the addition of lectin was largely affected by the degree of polymerization of DODA-PMEGal incorporated in the liposome (Figure 3). As for liposomes consisting of galactose lipids with a small DP (DODA-L-Gal has only one galactose residue), the recognition of the lipids by RCA is sterically not so easy, which results in a small value of the rate of turbidity change. With the increase in DP, the steric hindrance becomes smaller and the rate of turbidity change becomes larger (DP = 6.2). In the case of liposomes carrying galactose lipids with a large DP value (DP = 10, 15) the RCA molecule is captured in galactose-carrying long polymer chains on the liposome surface, and it is not so easy for RCA to bind to galactose residues on another liposome surface simultaneously, which reduces the rate of turbidity change. A similar tendency was previously observed in the recognition of glucose residues on the liposome surface by Concanavalin A (10).These results show the importance of steric hindrance for the recognition of sugar residues on the liposome surface by lectin molecules. The galactose-carrying novel amphiphiles, DODAPMEGal, can be easily prepared, and the degree of polymerization can be easily controlled by the ratio of the initiator (DODAdOl) and monomer (MEGal) used for polymerization. Therefore, the compounds prepared here would be highly useful as a tool for drug delivery system to hepatocytes. ACKNOWLEDGMENT
We are grateful to Wako Pure Chemicals for their kind donation of V-501. We wish to thank Professor S. Matsumura, Keio University, Yokohama, Japan, for his helpful suggestions for the preparation of MEGal. This work was supported by a Grant-in-Aid (06453153) from the Ministry of Education, Science and Culture. LITERATURE CITED
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134 Bioconjugate Chem., Vol. 6, No. 1, 1995
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