Synthesis and assembly of poly(ethylene glycol) - Lipids with mono-, di-, and tetraacyl chains and a poly(ethylene glycol) chain of various molecular weights

Article abstract of DOI:10.1021/ja000835+

We synthesized a series of amphiphiles with poly(ethylene glycol) [MW 2000 (PEG20), 5000 (PEG50), 12 500 (PEG125)] as a headgroup and one, two, or four palmitoyl chains (1C16, 2C16, or 4C16), using a lysine monodendron as a connector. The relationship between the hydrophilic - hydrophobic balance of the multiacyl PEG-lipids and the physicochemical characteristics in self- or co-assembly with phospholipids were studied. The PEG-lipids were easily synthesized by combination of a general liquid-phase peptide synthesis and the acylation of an amino acid. The PEG part of the PEG - lipid films was crystallized to show a typical spherulite pattern. The thermal properties and microscopic observation revealed the phase separation of PEG and acyl chain parts. The critical micelle concentrations (cmcs) mainly depend on the number of acyl chains rather than the molecular weight of the PEG chain, although the area per molecule is dependent on the molecular weight of the PEG chain rather than the number of the acyl chains. The gel-to-liquid crystalline phase transition temperature was increased with the increasing number of acyl chains and the decreasing molecular weight of the PEG chain. The PEG - lipids in the aqueous dispersions assemble to take fibrous structures with bimolecular thickness because of the intermolecular hydrogen bonding. The PEG - lipids were immobilized onto the surface of the phospholipid vesicles by simply adding their aqueous dispersions to the vesicle dispersion; however, they dissociated from the vesicles on dilution of the mixed dispersion because they were incorporated into the vesicles in an equilibrium state. To prevent the dissociation of the PEG - lipids, at least two and four acyl chains were required for PEG with M_W 5000 and 12 500, respectively. The aggregation of the vesicles by the addition of water-soluble polymers was significantly inhibited with the increasing molecular weight of the PEG chain. For the tight immobilization of the PEG - lipids with the long PEG chain onto the vesicular surface, an increased number of acyl chains is necessary, and the surface modification with the long PEG chains significantly increases the dispersion stability of the vesicles.

Full text of DOI:10.1021/ja000835+

J. Am. Chem. Soc. 2000, 122, 7927-7935
7927
Synthesis and Assembly of Poly(ethylene glycol)-Lipids with Mono-,
Di-, and Tetraacyl Chains and a Poly(ethylene glycol) Chain of
Various Molecular Weights
Shinji Takeoka, Katsura Mori, Haruki Ohkawa, Keitaro Sou, and Eishun Tsuchida*
Contribution from the Department of Polymer Chemistry, AdVanced Research Institute for Science and
Engineering, Waseda UniVersity, Tokyo 169-8555, Japan
ReceiVed March 7, 2000
Abstract: We synthesized a series of amphiphiles with poly(ethylene glycol) [MW 2000 (PEG20), 5000 (PEG50),
12 500 (PEG125)] as a headgroup and one, two, or four palmitoyl chains (1C16, 2C16, or 4C16), using a
lysine monodendron as a connector. The relationship between the hydrophilic-hydrophobic balance of the
multiacyl PEG-lipids and the physicochemical characteristics in self- or co-assembly with phospholipids were
studied. The PEG-lipids were easily synthesized by combination of a general liquid-phase peptide synthesis
and the acylation of an amino acid. The PEG part of the PEG-lipid films was crystallized to show a typical
spherulite pattern. The thermal properties and microscopic observation revealed the phase separation of PEG
and acyl chain parts. The critical micelle concentrations (cmcs) mainly depend on the number of acyl chains
rather than the molecular weight of the PEG chain, although the area per molecule is dependent on the molecular
weight of the PEG chain rather than the number of the acyl chains. The gel-to-liquid crystalline phase transition
temperature was increased with the increasing number of acyl chains and the decreasing molecular weight of
the PEG chain. The PEG-lipids in the aqueous dispersions assemble to take fibrous structures with bimolecular
thickness because of the intermolecular hydrogen bonding. The PEG-lipids were immobilized onto the surface
of the phospholipid vesicles by simply adding their aqueous dispersions to the vesicle dispersion; however,
they dissociated from the vesicles on dilution of the mixed dispersion because they were incorporated into the
vesicles in an equilibrium state. To prevent the dissociation of the PEG-lipids, at least two and four acyl
chains were required for PEG with Mw 5000 and 12 500, respectively. The aggregation of the vesicles by the
addition of water-soluble polymers was significantly inhibited with the increasing molecular weight of the
PEG chain. For the tight immobilization of the PEG-lipids with the long PEG chain onto the vesicular surface,
an increased number of acyl chains is necessary, and the surface modification with the long PEG chains
significantly increases the dispersion stability of the vesicles.
Introduction
surface to prolong the blood circulation time or to stabilize their
dispersion states.3-6 However, their cmcs are relatively high
(>10-5 M) because of the large headgroup, and the interbilayer
transfer rates of the PEG-phospholipids are also very fast
compared with those of phospholipids.7 In general, the molecular
weights of the PEG chain are between 2000 and 5000, and the
hydrophobic parts are usually taken from two choices: 1,2-
dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) or
1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE).
Though there are many papers on the molecular weight of PEG,
the hydrophobic part has not been paid much attention. We
clarified that the PEG-phospholipids were incorporated into
the bilayer membrane at an equilibrium state and removable
from the membrane by simple dilution.8 This result indicates
that the hydrophobic group of the PEG-phospholipids is not
large enough to anchor the PEG chain to the membrane.
Therefore, we designed macromolecular amphiphiles to im-
mobilize water-soluble polymers or proteins on the membrane
as illustrated in Figure 1. Our goals in designing such macro-
molecular amphiphiles are (1) simple syntheses, (2) chemically
The hydrophilic-hydrophobic balance of amphiphiles is the
most important factor in characterizing their physicochemical
properties.1 In biological systems, phospholipids with a small
hydrophilic headgroup, such as a glycerophosphocholine group,
assemble to form a bilayer membrane by hydrophobic interac-
tion among the diacyl chains with a carbon number of 16 or
18. The critical micelle concentrations (cmcs) of the phospho-
lipids are so small (<10-10 M) that the concentrations of free
monomers are negligible. However, in the case of the am-
phiphiles such as glycoproteins with a large hydrophilic
headgroup, their large headgroup can be immobilized in the
bilayer membrane with a large hydrophobic polypeptide as a
membrane-spanning anchor.2 The poly(ethylene glycol) (PEG)-
conjugated phosphatidylethanolamines (PEG-phospholipids)
have been widely used for the modification of the vesicular
* To whom all correspondence should be addressed.
(1) Israelachvili, J. N. Intermolecular and Surface Forces: With Ap-
plications to Colloidal and Biological Systems; Academic: London, 1985.
(2) Marchesi, V. T.; Furthmayr, H.; Tomita, M. Annu. ReV. Biochem.
1976, 45, 667.
(3) Klibanov, A. L.; Maruyama, K.; Torchilin, V. P.; Huang L. FEBS
Lett. 1990, 268, 235.
(5) Zalipsky, S. Bioconjugate Chem. 1995, 6, 150.
(6) Lasic. D. D.; Needham, D. Chem. ReV. 1995, 8, 2601.
(7) Silvius, J. R.; Zuckermann, M. J. Biochemistry 1993, 32, 3153.
(8) Sou, K.; Endo, T.; Takeoka, S.; Tsuchida, E. Bioconjugate Chem.
2000, 11, 372.
(4) Woodle, M. C.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1113,
171.
10.1021/ja000835+ CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/06/2000

7928 J. Am. Chem. Soc., Vol. 122, No. 33, 2000
Takeoka et al.
weight of PEG, n is the number of acyl chains, k is the carbon
number of the acyl chains) in solid and liquid states. Further-
more, the co-assembling properties of the PEG-lipids in a
mixed state with phospholipid vesicles were clarified in relation
to the structure of the PEG-lipids.
Experimental Section
Materials. L-Lysine, p-toluenesulfonic acid, benzyl alcohol (Bzl-
OH), and palmitic acid were purchased from Kanto Chemical Co.
R-Methyl-ω-aminomethylpoly(oxyethylene)s [Mn; 2050, 5050, 12 500,
Mw/Mn <1.03 gel permeation chromatography (GPC)] were purchased
from NOF Co. The mixed lipid powders of 1,2-dipalmitoyl-sn-glycero-
3-phosphatidylcholine (DPPC), cholesterol, and 1,2-dipalmitoyl-sn-
glycero-3-phosphatidylglycerol (DPPG) at 5:5:1 molar ratio (Presome
PPG-I) were purchased from Nippon Fine Chemical Co.
Figure 1. The concept to immobilize the large water-soluble polymers
on the bilayer vesicles. The PEG-lipids are spontaneously incorporated
into the bilayer membrane. The large number of acyl chains would be
necessary to immobilize the PEG chain with large molecular weight.
(a) mono-, (b) di-, and (c) tetraacyl PEG-lipids.
General Methods and Procedures. Melting points were determined
with a Seiko 120 DSC thermal analysis unit. The infrared (IR) spectra
were measured for the films and aqueous dispersions. The film was
prepared by casting the CHCl3 solution of the PEG-lipid on the KBr
plate. For the aqueous dispersion, the PEG-lipids were dispersed into
pure water at 60 °C. The sample dispersions were sandwiched between
the CaF2 windows, and the IR spectra were recorded on a JASCO FT-
IR-410. The 1H and 13C nuclear magnetic resonance (NMR) spectra of
solutions in CDCl3 or d6-DMSO were recorded on a JEOL JNM-LA500
(500 MHz) spectrometer. Fast-atom bombardment mass spectroscopy
(FAB-MS) spectra were obtained with a JEOL JMS-SX102A spec-
trometer. Analytical thin-layer chromatography (TLC) was performed
on commercial plates coated with silica gel (TLC MERK, silica gel 60
F254). The silica for flash chromatography was Merck Kieselgel 60
(230-400 mesh). The differential scanning calorimetry (DSC) analysis
was performed with a Seiko 120 DSC from 10 to 90 °C at a scan rate
of 2 °C min-1. The aqueous dispersion of the sample ([PEG-lipid] )
15 mM, 60 µL) was sealed in a silver pan. The compounds dissolved
in chloroform ([PEG-lipid] ) 10-3 kg dm-3) were cast on a glass
plate and observed with a polarized optical microscope.
distinct structures, (3) an appropriate hydrophilic-hydrophobic
balance for immobilization, (4) a parallel orientation of the acyl
chains not to disturb the orientation of phospholipids in bilayer
membrane, and (5) biocompatibility and biodegradability.
Dendrons, which constitute one part of a dendrimer and have
functional groups such as amino, hydroxy, or carboxylic acid
groups at the ends, are potent candidates to bind plural
molecules, and the number of the functional groups is control-
lable by changing the generation of the dendrons.9-14 A peptide
dendrimer based on lysine has been used to bind the saccha-
rides15 or peptides16,17 and is considered to be suitable for
biomaterials because it should be biocompatiable and biode-
gradable. An amphiphilic lysine dendrimer,18
a PEG-
Syntheses. The PEG-lipids were synthesized by a liquid-phase
dendrimer,19-21 and others have been reported, and the properties
at the air-water interface or aqueous phase were studied.22-25
We use the branched amino end groups and the carboxylic acid
group of lysine monodendrons to bind palmitoyl chains and a
R-methyl-ω-aminomethylpoly(oxyethylene) chain, respectively,
to synthesize PEG-lipids.
peptide synthetic method.18,26
PEG50-1C16. Palmitic acid (51 mg, 0.2 mmol) and N, N′-
dicyclohexylcarbodiimide (DCC; 41 mg, 0.2 mmol) were dissolved in
chloroform and stirred for 1 h at 4 °C. The solution was dropped into
a chloroform solution of PEG50-NH2 (0.5 g, 0.1 mmol) and (dimethyl-
amino)pyridine (DMAP) (24 mg, 0.2 mmol). The reaction was
examined by TLC (silica gel plate, chloroform/methanol ) 4/1, v/v),
and the PEG50-NH2 spot had completely disappeared after 2 h. The
product reprecipitated from ether was further purified by column
chromatography (silica gel, chloroform/methanol ) 6/1, v/v). The
PEG50-1C16 was obtained as a white powder (0.45 g, yield 86%).
TLC (silica gel) chloroform/methanol (4/1): Rf 0.70. IR (film; cm-1):
3375 (VN-H, amide), 1671 (VCdO, amide), 1540 (δN-H, amide);
1H NMR (CDCl3, 500 MHz, δ ppm): 0.88 (t, 3H, -CH3), 1.25 (s,
24H, -CH2-), 1.68 (m, 2H, -CH2-C-NH-), 1.84 (t, 2H, -CO-
C-CH2), 2.46 (t, 2H, -CO-CH2-), 2.46 (t, 2H, -CO-CH2-), 3.38
(s, 3H, -OCH3), 3.46 (t, 2H, -N-CH2), 3.64 (PEG). 13C NMR (CDCl3,
500 MHz, δ ppm): 14.13, 22.69, 29.36, 29.66, 31.93, 59.03, 70.58,
71.82. Anal. Calcd for C246H493N1O115: C 55.52, H 9.34, N 0.26;
found: C 55.51, H 9.61, N 0.53.
In this paper, we report on the syntheses and properties of a
series of the PEG-lipids (PEGm-nCk, m•102 is the molecular
(9) Tomalia, D. A. Sci. Am. 1995, 62.
(10) Moors, J. I. R.; Vogtle, F. Angew. Chem., Int. Ed. Engl. 1994, 33,
2431.
(11) Frechet, J. M. Science 1994, 263, 1710.
(12) Moore, J. S. Curr. Opin. Coll. Int. Sci. 1996, 1, 777.
(13) Narayanan, V. V.; Newkome, G. R. Top. Curr. Chem. 1998, 197,
19.
(14) Bosman, A. W.; Janssen H. M.; Meijer, E. W. Chem. ReV. 1999,
99, 1665.
(15) Zanini, D.; Roy, R. Bioconjugate Chem. 1997, 8, 187.
(16) Posnett, D. N.; McGrath, H.; Tam, J. P. J. Biol. Chem. 1988, 263,
1719.
(17) Pessi, A.; Bianchi, E.; Bonelli, F.; Chiappinelli, L. J. Chem. Soc.
Chem. Commun. 1990, 8.
(18) Chapman, T. M.; Hillyer, G. L.; Mahan, E. J.; Shaffer, K. A. J.
Am. Chem. Soc. 1994, 116, 11195.
(19) Gitsov, I.; Wooley, K. L.; Hawker, C. J.; Ivanova, P. T.; Frechet,
M. J. Macromolecules 1993, 26, 5621.
(20) Gitsov, I.; Frechet, M. J. Macromolecules 1993, 26, 6536.
(21) Gitosov, I.; Frechet, J. M. J. J. Am. Chem. Soc. 1996, 118, 3785.
(22) Saville, P. M.; White, J. W.; Hawker, C. J.; Wooley, K. L.; Frechet,
J. M. J. J. Phys. Chem. 1993, 97, 293.
(23) van Hest, J. C. M.; Baars, M. W. P. L.; Elissen-Roman, C.; van
Genderen, M. H. P.; Meijer, E. M. Macromolecules 1995, 28, 6689.
(24) Schenning, A. P. H. J.; Elissen-Roman, C.; Weener, J.-W.; Baars,
M. W. P. L.; van der Gaast, S. J.; Meijer, E. W. J. Am. Chem. Soc. 1998,
120, 8199.
Bis-palmitoyl-L-Lys. The L-lysine (Lys; 5.1 g, 35.2 mmol) and
p-toluenesulfonic acid (monohydrate, 14.7 g, 77.3 mmol) were dissolved
into benzyl alcohol (Bzl-OH; 14.0 g, 124.1 mmol) and benzene (30
mL).27 The mixture was refluxed for 6 h at 100 °C with trapping
generated water with a Dean-Stark receiver. The reaction mixture was
allowed to cool to room temperature and reprecipitated three times with
(26) The first poly(L-lysine) dendrimer: Denkewalter, R. G.; Kolc, J.;
Lukasavage, W. J. U.S. Patent 4, 289, 872, Sept 15, 1981. (b) The Choi, J.
S.; Joo, D. K.; Kim, C. H.; Kim, K.; Park, J. S. J. Am. Chem. Soc. 2000,
122, 474. (c) A solid-phase synthesis of poly(L-lysine) dendrimer: Roy,
R.; Zanini, D.; Meunier, S. J.; Romanowska, A. J. Chem. Soc. Chem.
Commun. 1993, 1869.
(25) Frechet, J. M.; Gitosov, I.; Monteil, T.; Rochat, S.; Sassi, J. F.;
Vergelati, C.; Yu, D. Chem. Mater. 1999, 11, 1267.
(27) Zervas, L.; Winitz, M.; Greenstein, J. P. J. Org. Chem. 1955, 22,
1515.

Synthesis and Assembly of Various Poly(ethylene glycol)-Lipids
J. Am. Chem. Soc., Vol. 122, No. 33, 2000 7929
1
ether (200 mL) at 4 °C. After recrystallization from methanol/ether at
4 °C, L-Lys-O-Bzl (TsOH)2 was obtained as a white solid (18.0 g, yield
88%).
amide), 1556 (δN-H, amide). H NMR (CDCl3, 500 MHz, δ ppm):
0.88 (t, 6H, -CH3), 1.25 (s, 50H, -CH2-CH2-, Lys γ-CH2), 1.32
(m, 2H, Lys δ-CH2), 1.63-1.80 (br, 8H, -CH2-C-N-, -N-CO-
C-CH2-, Lys â-CH2), 2.27, 2.38 (t, 4H, -N-CO-CH2-), 3.29 (m,
2H, Lys ꢀ-CH2), 3.38 (s, 3H, -O-CH3), 3.43 (br, 2H, -CH2-NH-),
3.66 (PEG), 4.39 (m, 1H, Lys R-CH). 13C NMR (CDCl3, 500 MHz, δ
ppm): 14.12, 22.68, 25.74, 28.75, 29.34, 29.53, 29.65, 31.92, 36.35,
38.13, 59.02, 70.44, 71.95.
The palmitic acid (3.2 g, 12.4 mmol) and DCC (2.6 g, 12.4 mmol)
were dissolved in chloroform and stirred for 1 h at 4 °C. The L-Lys-
O-Bzl(TsOH)2 (3.0 g, 5.12 mmol) and DMAP (1.4 g, 11.4 mmol) were
added to the mixture and stirred at 4 °C for 14 h and then at 25 °C for
2 h. The crude product was washed with a Na2CO3 saturated aq.
solution. After recrystallization from methanol at 4 °C, bis-palmitoyl-
L-Lys-O-Bzl was obtained as a white solid (2.9 g, yield 79%).
Bis-palmitoyl-L-Lys-OBzl (1.52 g, 2.13 mmol) was dissolved in a
mixed solvent (43 mL, chloroform/methanol ) 10/7, v/v), and a 1 N
NaOH solution (3.4 mL) was added and stirred for 4 h at 25 °C. The
1 N HCl solution was added to the solution until a pH of 3.0 was
attained. The product was washed with water and then methanol. Bis-
palmitoyl-L-Lys (1.30 g, yield 98%) was obtained as a white solid.
TLC (silica gel) chloroform/methanol (4/1): Rf 0.45. IR (film; cm-1):
3305 (VN-H, amide), 1721 (VCdO, carbonyl), 1638 (VCdO, amide),
PEG125-2C16. Yield 61%. TLC (silica gel) chloroform/methanol
(4/1): Rf 0.63. IR (film; cm-1): 3305 (VN-H, amide), 1638 (VCdO,
1
amide), 1556 (δN-H, amide). H NMR (CDCl3, 500 MHz, δ ppm):
0.88 (t, 6H, -CH3), 1.25 (s, 50H, -CH2-CH2-, Lys γ-CH2), 1.32
(m, 2H, Lys δ-CH2), 1.63-1.80 (br, 8H, -CH2-C-N-, -N-CO-
C-CH2-, Lys â-CH2), 2.27, 2.38 (t, 4H, -N-CO-CH2-), 3.29 (m,
2H, Lys ꢀ-CH2), 3.38 (s, 3H, -O-CH3), 3.43 (br, 2H, -CH2-NH-),
3.66 (PEG), 4.39 (m, 1H, Lys R-CH). 13C NMR (CDCl3, 500 MHz, δ
ppm): 14.12, 22.68, 25.74, 28.75, 29.34, 29.53, 29.65, 31.92, 36.35,
38.13, 59.02, 70.44, 71.95.
1
PEG50-4C16. Bis-palmitoyl-L-Lys (100 mg, 0.15 mmol) and DCC
(30 mg, 0.15 mmol) were dissolved in chloroform and stirred for 1 h
at 4 °C. The reaction mixture was added to a PEG50-L-Lys (200 mg,
0.038 mmol) and DMAP (4.5 mg, 0.037 mmol) chloroform solution
and stirred for 6 h at 25 °C. The reaction mixture was filtered with a
glass filter, and the filtrate was reprecipitated from ether. The crude
product was further purified by column chromatography (silica gel plate,
solvent: chloroform/methanol ) 6/1, v/v). The PEG50-4C16 was
obtained as a white powder (180 mg, yield 78%). TLC (silica gel)
chloroform/methanol (4/1): Rf 0.78. IR (film; cm-1): 3305 (VN-H,
amide), 1637 (VCdO, amide), 1560 (δN-H, amide). 1H NMR (CDCl3,
500 MHz, δ ppm): 0.88 (t, 12H, -CH3), 1.25 (s, 110H, -CH2-CH2-,
Lys γ-CH2), 1.35-1.90 (br, 22H, Lys δ-CH2, -CH2-C-N-, -N-
CO-C-CH2-, Lys â-CH2), 2.00-2.45 (br, 8H, -N-CO-CH2-),
3.30 (br, 6H, Lys ꢀ-CH2), 3.38 (s, 3H, -O-CH3), 3.43 (br, 2H, -CH2-
NH-), 3.66 (PEG), 4.48 (br, 3H, Lys R-CH2). 13C NMR (CDCl3, 500
MHz, δ ppm): 14.12, 22.68, 25.79, 26.15, 29.36, 29.42, 29.59, 29.72,
31.93, 59.03, 70.58, 71.95. Anal. Calcd for C312H619N7O121: C 58.51,
H 9.74, N 1.53; found C 58.20, H 10.04, N 1.7.
1553 (δN-H, amide); H NMR (DMSO-d6, 500 MHz, δ ppm): 0.84
(t, 6H, -CH3), 1.24 (s, 50H, -CH2-CH2-, Lys γ-CH2), 1.36 (m, 2H,
Lys δ-CH2), 1.47 (m, 4H, -N-CO-C-CH2-), 1.55, 1.67 (m, 2H,
Lys â-CH2), 2.02, 2.09 (t, 4H, -N-CO-CH2-), 2.99 (m, 2H, Lys
ꢀ-CH2), 4.14 (m, 1H, Lys R-CH), 7.55 (br, 1H, -NH-CO-), 7.78 (d,
1H, -NH-CO-), 12.23 (br, 1H, -COOH). MS (FAB): calcd for
C38H74N2O4: 623.0; found: 623.5 (M+H)+. Anal. Calcd for
C38H74N2O4: C 73.26, H 11.97, N 4.50; found C 72.39, H 12.43, N
4.74.
PEG-L-Lys. The L-Lys (1.5 g, 10.3 mmol) and t-buthoxyoxycarbonyl
anhydride (6.3 g, 29.0 mmol) were dissolved into a mixed solvent of
dioxan (10 mL), water (10 mL), and 1 N NaOH (10 mL) and stirred
for 6 h at pH 7.3, and 25 °C. The reaction mixture was concentrated
up to 10 mL, and pH was adjusted to 2.4 by adding a KHSO4 aq.
solution. The product was extracted with ethyl acetate and reprecipitated
from hexane. The L-Lys-(Boc)2 was obtained as a white solid (2.64 g,
yield 74%).
The L-Lys-(Boc)2 (83 mg, 0.24 mmol) and DCC (50 mg, 0.24 mmol)
were dissolved in chloroform and stirred for 1 h at 4 °C. The reaction
mixture was added to a PEG50-NH2 (1.0 g, 0.2 mmol) and DMAP (24
mg, 0.2 mmol) chloroform solution and stirred for 6 h at 4 °C. The
crude product was purified by reprecipitation from ether. The PEG-L-
Lys-(Boc)2 (0.97 g, yield 90%) was thus obtained as a white solid.
The PEG50-L-Lys-(Boc)2 (0.5 g, 0.09 mmol) was dissolved in
trifluoroacetic acid (5 mL) and stirred for 1 h at 4 °C. The product
was purified by reprecipitation from ether. The PEG50-L-Lys (0.44
g, yield 91%) was obtained as a white solid. TLC (silica gel)
The PEG125-4C16 was obtained as a white powder (yield: 77%)
by the same method. TLC (silica gel) chloroform/methanol (4/1): Rf
0.68. IR (film; cm-1): 3292 (VN-H, amide), 1635 (VCdO, amide),
1
1552 (δN-H, amide). H NMR (CDCl3, 500 MHz, δ ppm): 0.88 (t,
12H, -CH3), 1.25 (s, 110H, -CH2-CH2-, Lys γ-CH2), 1.35-1.90
(br, 22H, Lys δ-CH2, -CH2-C-N-, -N-CO-C-CH2-, Lys
â-CH2), 2.00-2.45 (br, 8H, -N-CO-CH2-), 3.30 (br, 6H, Lys
ꢀ-CH2), 3.38 (s, 3H, -O-CH3), 3.43 (br, 2H, -CH2-NH-), 3.66
(PEG), 4.48 (br, 3H, Lys R-CH2). 13C NMR (CDCl3, 500 MHz, δ
ppm): 14.12, 22.68, 25.79, 26.15, 29.36, 29.42, 29.59, 29.72, 31.93,
59.03, 70.58, 71.95.
1
chloroform/methanol (4/1): Rf 0.32. H NMR (CDCl3, 500 MHz, δ
ppm): 1.60 (m, 2H, Lys γ-CH2), 1.80-1.90 (m, 4H, -CH2-C-N-,
Lys δ-CH2), 2.05 (m, 2H, Lys â-CH2), 3.06 (t, 2H, -CH2-N-CO-),
3.25-3.40 (m, 2H, Lys ꢀ-CH2), 3.38 (s, 3H, -O-CH3), 3.65 (PEG),
4.15 (m, 1H, Lys R-CH), 7.64, 7.83, 8.41 (br, 7H, -NH-CO-,
-NH3+).
Critical Micelle Concentration (cmc). The cmc was measured by
an iodine solubilization method.28 The PEG-lipids with different
amounts were dispersed into an iodine aqueous solution ([I2] ) 30 mg
L-1). The transmittance at the maximum absorption wavelength of 360
nm was measured with a UV-vis spectrophotometer (MPS-2000,
Shimadzu Co.). The relationship between the concentration of the
PEG-lipids and the logarithm of the transmittance was used to
determine the cmc from the inflection point of the relation.
Langmuir-Blodgett Experiment. A monolayer of the PEG-lipids
was spread from a portion (7 or 20 µL) of the chloroform solutions
(0.5 mM) of the PEG-lipids on the subphase of pure water at 25 °C.
The surface area was varied by moving a Teflon-coated barrier
automatically at a rate of 28 cm2 min-1 in a thermostated Langmuir
trough (HBM-type balancemeter; Kyowa Interface Science Co.). The
surface pressure was measured by a Wilhelmy-plate method.29 The
surface pressure and total surface area were recorded with an X-Y
recorder.
PEG50-2C16. Bis-palmitoyl-L-Lys (125 mg, 0.2 mmol) and DCC
(41 mg, 0.2 mmol) were dissolved in chloroform and stirred for 30
min at 25 °C. PEG50-NH2 (0.5 g, 0.1 mmol) and DMAP (24 mg, 0.2
mmol) were added to the mixture and stirred at 25 °C for 6 h. The
product reprecipitated from ether was further purified by column
chromatography (silica gel, chloroform/methanol ) 6/1, v/v). The
PEG50-2C16 was obtained as a white powder (500 mg, yield; 88%).
TLC (silica gel) chloroform/methanol (4/1): Rf 0.73. IR (film; cm-1):
3294 (VN-H, amide), 1634 (VCdO, amide), 1553 (δN-H, amide);
1H NMR (CDCl3, 500 MHz, δ ppm): 0.88 (t, 6H, -CH3), 1.25 (s,
50H, -CH2-CH2-, Lys γ-CH2), 1.32 (m, 2H, Lys δ-CH2), 1.63-
1.80 (br, 8H, -CH2-C-N-, -N-CO-C-CH2-, Lys â-CH2), 2.27,
2.38 (t, 4H, -N-CO-CH2-), 3.29 (m, 2H, Lys ꢀ-CH2), 3.38 (s, 3H,
-O-CH3), 3.43 (br, 2H, -CH2-NH-), 3.66 (PEG), 4.39 (m, 1H, Lys
R-CH). 13C NMR (CDCl3, 500 MHz, δ ppm): 14.12, 22.68, 25.74,
28.75, 29.34, 29.53, 29.65, 31.92, 36.35, 38.13, 59.02, 70.44, 71.95.
Anal. Calcd for C268H535N3O117: C 56.59, H 9.48, N 0.74; found C
56.88, H 9.87, N 1.07.
Transmission Electron Microscopy (TEM). To prepare samples
for TEM, the PEG-lipids were dispersed in pure water at 60 °C. One
(28) Ross, S.; Oliver, J. P. J. Phys. Chem. 1959, 63, 1671.
(29) Fendler, J. H. Membrane Mimetic Chemistry: Characterization and
Applications of Micelles, Microemulsions, Monolayers, Bilayers, Vesicles,
Host-Guest Systems, and Polyions; John Wiley & Sons: New York, 1982.
PEG20-2C16s. Yield 60%. TLC (silica gel) chloroform/methanol
(4/1): Rf 0.75. IR (film; cm-1): 3311 (VN-H, amide), 1637 (VCdO,

7930 J. Am. Chem. Soc., Vol. 122, No. 33, 2000
Scheme 1. Synthesis of the PEG-Lipids
Takeoka et al.
drop of the dispersion was dropped onto a carbon-coated copper grid,
and then the excess solution was immediately removed with the help
of a filter paper. One drop of an aqueous solution of sodium
phosphotungstate (Kanto Chemical Co.; 0.01 kg dm-3) was then
dropped on the grid and treated as just described. The grid, kept in a
desiccator for 12 h, was observed with a TEM (JEM-1010, JEOL Co.)
using an acceleration voltage of 100 kV.
Fluorescence Depolarization Method. For the fluorescence depo-
larization method, a 1,6-diphenyl-1,3,5-hexatriene (DPH) solution [5
µL, [DPH] ) 0.5 mM, in tetrahydrofuran (THF)] was added to a
dispersion of the PEG-lipids (2.5 mL, [PEG-lipids] ) 1 mM). A
THF solution in the absence of DPH was added to the dispersion of
the PEG-lipids as a blank. These samples were incubated at 37 °C
for 2 h. Fluorescence spectroscopy was carried out with a JASCO FP-
770 spectrofluorometer equipped with polarizers. DPH was excited at
357 nm and then detected at 430 nm.
determine the incorporated ratio of the PEG-lipids.8 The same
measurement was carried out for the control samples, where unincor-
porated PEG-lipids were not removed, to determine the peak area ratio
(R0) of the choline methyl proton of DPPC to the methylene proton of
PEG for the total amount of the PEG-lipids. The incorporation ratio
was calculated from a percentage of R1 to R0.
Critical Molecular Weight (Mc). To determine a critical molecular
weight (Mc) of water-soluble polymers for the aggregation of vesicles,
we used plain PEG with various molecular weights. We added a solution
of the PEGs (500 µL, 0.15 kg dm-3) to the vesicle dispersion (2 mL,
1.39 mM) in a cuvette (l ) 1 cm) at 37 °C. The turbidity change (∆OD)
at 600 nm was monitored 15 min after the addition of PEGs with a
UV-vis spectrophotometer (MPS-2000, Shimadzu Co.).31 The Mc was
determined as the minimum molecular weight of the PEGs for the
initiation of turbidity increase.
Preparation of PEG-Incorporated Vesicles. Mixed lipids were
dissolved in chloroform and dried in vacuo to form a film on the wall
of a flask. After the film was hydrated and dispersed into a Tris-HCl
buffer solution ([Tris] ) 20 mM, [NaCl] ) 140 mM, pH 7.4) with
glass beads, it was extruded through polycarbonate membrane filters
(the final pore size of the filter was 0.2 µm). The diameter of the
resulting vesicles was determined to be 250 ( 40 nm with a COULTER
submicron particle analyzer (Coulter, Hialeah, FL). The total surface
area of the vesicles in the dispersion was measured with 6-p-toluidino-
2-naphthalenesulfonic acid (Tns),30 and the average number of bilayer
membranes of the vesicle was calculated to be 2.1.
A dispersion of the PEG-lipids (19 mL, [PEG-lipids] ) 17.5 µM)
was added to the vesicle dispersion (25 mL, [lipids] ) 17.3 mM) and
stirred at 37 °C. The incorporation of the PEG-lipids into the surface
of the vesicles was continuously monitored as a thermal change with
an isothermal titration microcalorimeter (MCS ITC, Microcal, Inc.).
For the measurement of the incorporation ratio, the sample was
centrifuged (100 000 g, 60 min) at 37 °C to remove the unincorporated
PEG-lipids as a supernatant. The precipitate was freeze-dried and
dissolved in CDCl3. The peak area ratio (R1) of the choline methyl
proton of DPPC (3.39 ppm) to the methylene proton of PEG (3.63
ppm) was measured by 1H NMR spectroscopy (JEOL JNM-LA500) to
Results and Discussion
Syntheses of PEG-Lipids. We synthesized a series of
PEGm-nCk compounds with PEG [MW 2000 (PEG20), 5000
(PEG50), 12 500 (PEG125)] as a headgroup and one, two, or
four palmitoyl chains (1C16, 2C16, or 4C16) as shown in
Scheme 1. The PEGm-1Ck and PEGm-2Ck could be quite
easily synthesized by coupling the PEGm with 1Ck and 2Ck,
respectively. The PEGm-4Ck was also easily synthesized, even
if a reaction step to couple the lysine protected by t-butoxy-
oxycarbonyl groups was added. The yields of those compounds
were high (∼80%).
Our synthetic route can be generalized as follows. A water-
soluble polymer (P-B) with a reactive functional group (B) at
one end is coupled with the functional group A of a monomer
[A-2(BC)], which also has two functional groups B protected
by C. After removing C, the monodendron structure can be built
at the end of the P by repeating the coupling reaction of B with
A of the other monomers. Finally, the di-acyl derivatives
[A-2(BD)] are coupled with the end groups B. The number of
(30) Takeoka, S.; Ohgushi, T.; Terase, K.; Ohmori, T.; Tsuchida, E.
Langmuir 1996, 12, 1775.
(31) Takeoka, S.; Sou, K.; Arase, S.; Ohgushi, T.; Tsuchida, E.
Macromolecules, 1996, 29, 8132.

Synthesis and Assembly of Various Poly(ethylene glycol)-Lipids
Table 1. Melting Points of the PEGm-nCk Amphiphiles
J. Am. Chem. Soc., Vol. 122, No. 33, 2000 7931
the cmc of the PEG-lipids because of the polymer effect. There
are many papers on the properties of monoalkyl-PEGs, including
the relationship between the molecular weight of the PEG part
and the cmcs of those surfactants.34-36 The increment rate of
the cmc with the increasing degree of polymerization gradually
decreases when the degree of polymerization becomes high. This
phenomenon is the polymer effect; namely, a smaller effect of
the PEG molecular weight on the cmc when the molecular
weight becomes higher. The cmcs of PEG50-4C16 and
PEG125-4C16 were 26 and 25 µM, respectively. The cmc was
decreased with the increasing number of the acyl chains.
The cmcs of the PEG-lipids were generally quite high in
comparison with those of phospholipids. For instance the cmc
of DPPC is ∼10-10 M,37 and it jumps tremendously due to the
conjugation with hydrophilic PEG. For example, the cmc of
PEG50-DPPE is 70 µM (Tris buffer, pH 7.4, 37 °C),8 that of
PEG50-DSPE is 9 µM (Tris buffer, pH 7.4, 37 °C),8 and that
of PEG20-DSPE is 5.8 µM (saline).38 Of course, direct
comparison among these data is impossible because the
experimental conditions are not the same. The cmc roughly
depends on the length of the acyl chains. It should be noted
that based on the cmc, the PEG-lipids still have strong
hydrophilicity even if they have four acyl chains.
Monolayer Study. Figure 2 is the surface pressure (π)-
surface area (A) isothermal curves of monolayers of the PEG-
lipids and the lipids without PEG parts (1C16, 2C16, 4C16).
The areas per molecule are summarized in Table 2. As shown
in Figure 2(a), the area per molecule of the PEG-free lipids
clearly depends on the number of acyl chains. The areas per
molecule of 1C16, 2C16, and 4C16, calculated from the
extrapolation of the slope to zero surface pressure, were 0.21,
0.52, and 1.10 nm2, respectively. The limiting areas per molecule
were 0.20, 0.40, and 0.92 nm2 for 1C16, 2C16, and 4C16,
respectively. If the acyl chains of the PEG-lipid would order
in parallel with each other, the occupied areas of the acyl chains
for 1C16, 2C16, and 4C16 would be calculated from Corey-
Pauling-Koltun (CPK) models to be 0.2, 0.5, and 1.2 nm2,
respectively. These values show good agreement with the
observed ones. The di- or triacyl amphiphiles with two head-
groups (so-called “gemini surfactants”) were studied from the
π-A isotherms.39 The area per molecule of a triacyl amphiphile
was also larger than that of a diacyl one.
compound
Tm (°C)
∆H (kcal mol-1
)
PEG50-1C16
PEG20-2C16
PEG50-2C16
PEG125-2C16
PEG50-4C16
PEG125-4C16
2C16
59.5
211.0
47.0, 95.7
59.3, 86.4
62.4, 77.6
56.2, 154.7
61.5, 145.9
119.5
42.4, 18.1
199.8, 21.7
448.7, 13.2
164.8, 12.7
415.2, 6.9
24.4
125.4
4C16
121.1
159.4
9.3
12.5
PEG20
PEG50
PEG125
54.1
63.0
63.5
78.9
229.8
514.8
acyl chains is calculated to be 2n, where n is the generation
number of the monodendron. In the case of PEG-4C16, the
repeating number of the coupling reaction was 2 and n was 2.
Solid State Properties. The melting points are summarized
in Table 1. PEG50-1C16 has a single melting point at 59.5
°C. Interestingly, the other samples have two distinct melting
points; one melting point is near 60 °C, which can be the melting
point of the PEG chain [the melting point of pure PEG with a
high molecular weight (Mw > 5000) is 63 °C]. The melting
enthalpy increases with the molecular weight of PEG, and each
enthalpy corresponds to that of the plain PEG with the same
molecular weight. The other higher melting point increases with
the increasing number of acyl chains. When the number of acyl
chains is constant, the increasing molecular weight of the PEG
part results in a drop in the latter melting point. Therefore, the
latter melting point should be identified as the melting point of
the acyl chains. The number of acyl chains in one PEG-lipid
would increase the packing of the acyl chains, leading to the
enhancement of the intramolecular interaction of hydrogen
bonding, whereas the steric hindrance of the PEG part would
disturb the intermolecular packing of the acyl chains. The two
melting points indicate the phase-separation between the PEG
part and the acyl chain part. The melting behavior depends on
the ratio of the hydrophilic and hydrophobic parts of the PEG-
lipids, such as the block copolymers reported elsewhere.19,20
The spherulite structure, typical of crystallized PEG, was
observed with a polarized optical microscope, indicating that
the phase-separated PEG phase characterizes the morphology
of the PEG-lipid crystal. However, the diameter of the
spherulites of the PEG-lipids tends to be small compared with
that of pure PEG. Especially, a unique morphology band-like
texture was observed for PEG50-4C16, suggesting a different
orientation due to the influence of the bulky hydrophobic region.
This structure is assumed to be a lyotropic liquid crystal typically
observed for amphiphiles.32,33
Interestingly, the isotherms of the 2C16 and 4C16 reached a
limiting area without showing a clear collapsing point. This
result indicates that some cohesive force between those mol-
ecules would exist in the monolayer membrane to stabilize the
membrane. This force would be hydrogen bonding among the
amide groups of the lipids.
The monoacyl PEG-lipid (PEG50-1C16) shows no steep
increase in the surface pressure during compression, indicating
the solubilization of the PEG-lipid into the subphase, as shown
in Figure 2(b). The other PEG-lipids have an expanded liquid
membrane phase, which shows the gradual increase of the
surface pressure over the wide surface area. This phase should
be a process to compress the entangled PEG chain. Therefore,
Critical Micelle Concentration (cmc). PEG50-1C16 is
easily dispersed into pure water to become a transparent solution,
whereas PEG50-2C16 and PEG50-4C16 are dispersed with
difficulty even by heating above 45 and 60 °C, respectively.
Table 2 summarizes the cmcs of the synthesized PEG-lipids.
The cmc decreases with the increasing number of acyl chains
(i.e., with the enlargement of the hydrophobic part). There is
little difference in the cmcs between PEG50-2C16 (59 µM)
and PEG125-2C16 (63 µM); however, PEG20-2C16 has a
somewhat lower cmc (34 µM). From these results, the molecular
weight of PEGs with molecular weight >5000 would not affect
(34) Becher, P. J. Phys. Chem. 1959, 63, 1675.
(35) Shick, M. J.; Atlas, S. M.; Eirich, F. R. J. Phys. Chem. 1962, 66,
1326.
(36) Jonsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactants
and Polymers in Aqueous Solution, J. Wiley & Sons: Chechester, 1998; p
38.
(37) Smith, R.; Tanford C. J. Mol. Biol. 1972, 67, 75.
(38) Uster, P. S.; Allen, T. M.; Daniel, B. E.; Mendez, C. J.; Newman,
M. S.; Zhu, G. Z. FEBS Lett. 1996, 386, 243.
(32) Zhao, W.; Kloczkowski, A.; Mark, J. E.; Ermam, B.; Bahar, I.
Macromolecules 1996, 29, 2805.
(33) Alexandridis, P.; Olsson, U.; Lindmam, B. Langmuir, 1998, 14,
2627.
(39) Sumida, Y.; Oki, T.; Masuyama, A.; Maekawa, H.; Nishiura, M.;
Kida, T.; Nakatsuji, Y.; Ikeda, I.; Nijima, M. Langmuir 1998, 14, 7450.

7932 J. Am. Chem. Soc., Vol. 122, No. 33, 2000
Takeoka et al.
Table 2. Physicochemical Parameters of PEGm-nCk Amphiphiles in Aqueous Solution
compound
HLB
cmc (µM)a
area per molecule (nm2)
Tc (°C)b
∆H (kcal mol-1
)
frequency of amide I (cm-1
)
PEG50-1C16
PEG20-2C16
PEG50-2C16
PEG125-2C16
PEG50-4C16
PEG125-4C16
1C16
23.1
15.2
19.8
24.3
16.6
20.9
-
147
34
59
63
26
25
-
soluble
2.6c
non (L)
62
54
non (L)
non (G)
non (G)
-
-
-
non
2.8
2.0
non
non
non
-
1651
1641
1641
1645
1637
1642
-
5.4c
11.6c
8.2c
11.6c
0.21d
0.20e
0.40e
0.92e
2C16
4C16
-
-
0.52d
1.10d
-
-
-
-
-
-
a In pure water at 37 °C. b L, liquid crystalline phase; G, gel phase. c At 30 mN/m in pure water at 25 °C. d Area per molecule calculated from
extrapolation of slope to 0 mN/m. e Limited area per molecule.
lipids with molecular weights of PEG20, 50, and 125 were
calculated to be 3.5, 6.0, and 10.4 nm, respectively, and thus
the molecular areas of those lipids were estimated to be 9.6,
28.3, and 84.9 nm2, respectively. Therefore, the PEG chains
would not be freely entangled but be compressed with each other
to form a condensed structure.
Assembling Structures. Fibrous assembling structures were
observed by TEM for PEG20-2C16, PEG50-4C16, and
PEG125-4C16 with almost uniform diameters of 6, 5, and 5
nm, respectively. The standard deviation of the diameters was
within 1 nm. Considering the CPK models, the lengths of the
hydrophobic part of 2C16 and 4C16, including the dendron part,
were calculated to be 2.3 and 2.8 nm, respectively. The lengths
of the bilayer were thus estimated to be 4.6 and 5.6 nm,
respectively. Those values roughly agree with the negatively
stained parts of the objects obtained from the TEM photographs
(Figure 3). This result suggests that the base of the assembling
structure would be constructed from a bimolecular unit. In
addition, many fibers take parallel orientation, leaving spaces
of 4, 6, and 11 nm for PEG20, PEG50, and PEG125,
respectively, which are in good agreement with the RF values
of the PEG chains. This result indicates the interdigital arrange-
ment of the PEG entanglement between neighboring fibers.
Therefore, the stained space between the fibers in Figure 3 is
considered to be filled with PEG chains, which would prevent
further access to each other by steric hindrance. The staining
reagent, sodium phosphotungstate, should penetrate into the PEG
chain domain because PEG could solubilize this salt by strong
ion-dipole interaction.
The assembling structure of the PEG50-1C16 and PEG50-
Figure 2. Surface pressure (π)-surface area (A) isotherms of the (a)
nC16 and (b) PEGm-nC16 monolayer membranes at the air-water
interface at 25 °C.
2C16 seems to be a ribbonlike structure. The ribbons of the
PEG50-1C16 were frequently twisted, and the observed width
varied from 5 to 22 nm. This variation means that the ribbons
of PEG50-1C16 have a bilayer thickness because the minimum
width (5 nm) corresponds to the bimolecular layer of 1C16 (4.2
nm). On the other hand, the ribbons of PEG50-2C16 are rather
flat. The width varied from 14 to 36 nm. PEG125-2C16 has a
globular structure of 20 ( 5 nm diameter.
the PEG chain with a higher molecular weight shows a larger
surface pressure for the same molecular area.
A steep increase in the surface pressure was observed from
10 nm2 for PEG20, from 15 nm2 for PEG50, and from 20 nm2
for PEG125, independent of the number of acyl chains. This
increase represents the phase transition from the expanded liquid
membrane to the compressed solid one. However, the molecular
areas obtained by extrapolation from the steep increasing part
of the curves to the X-axis in Figure 2(b) were considerably
larger than the corresponding surface areas already calculated
and depicted in Table 2. This difference indicates that the
molecular area would be determined from the maximum cross-
sectional area of the entangled PEG chain of the PEG-lipids
as a typical characteristic of the amphiphiles with a flexible
linear chain as a headgroup. The expansion of the entangled
polymer chain in a good solvent can be calculated from the
Flory’s empirical equation as RF ) aN3/5, where RF is the inertial
diameter of the polymer entanglement.40 The RF of the PEG-
In general, amphiphilic molecules, of which the headgroup
is considerably large compared with the acyl chains, tend to
form globular micelles.1 For instance, PEG19-DSPE and
PEG50-DSPE are known to form micelles.4 In contrast, our
PEG-lipids tend to form fibrous structures. If the amphiphilic
molecule would have asymmetric carbon atoms or intermo-
lecular hydrogen bonding in addition to hydrophobic interaction
in assemblies, the amphiphile would orient to form fibers.41
Fourier Transform-Infrared (FT-IR) Measurement. The
band shift of the FT-IR spectra provides useful information about
(40) Kenworthy, A. K.; Hristova, K.; Needham, D.; McIntosh, T. J.
Biophys. J. 1995, 68, 1921.
(41) Fuhrhop, J.-H.; Schnieder, P.; Rosenberg, J.; Boekema, E. J. Am.
Chem. Soc. 1987, 109, 3387.

Synthesis and Assembly of Various Poly(ethylene glycol)-Lipids
J. Am. Chem. Soc., Vol. 122, No. 33, 2000 7933
Figure 3. Transmission electron micrographs of the self-assemblies of the PEGm-nC16 amphiphiles. The PEGm-nC16 amphiphiles were dispersed
in pure water at 60 °C.
hydrogen bonding.42,43 The amide I band of the free amide bond
is located at ∼1680 cm-1 and shifts to the lower wavenumber
depending on the strength of the hydrogen bonding. The amide
I band of the aqueous dispersions of the PEG-lipids appears
at 1651, 1641, 1641, 1645, 1637, and 1642 cm-1 for PEG50-
1C16, PEG20-2C16, PEG50-2C16, PEG125-2C16, PEG50-
4C16, and PEG125-4C16, respectively, as shown in Table 2.
The larger degree of deviation from the wavenumber of the free
amide bond indicates the stronger hydrogen bonding of the
PEG-lipids in assemblies. Therefore, PEG50-1C16 and
PEG125-2C16 have relatively weak hydrogen bonding, whereas
PEG50-4C16 has the strongest bonding. The strength of the
hydrogen bonding tends to increase with the increasing number
of the acyl chains and the lowering molecular weight of the
PEG chains. Therefore, our PEG-lipids, which have strong
hydrogen bonding at the lysine dendron part, would assume
fibrous structures rather than micelles.
mol-1) for PEG50-2C16. The other PEG-lipids did not show
any peaks in a temperature region from 5 to 90 °C. As a
reference, DPPC with the same chain length as the PEG-lipids
just mentioned, showed a phase transition temperature of 41
°C. Therefore, the lysine dendron structure would account for
the higher phase transition temperatures of the PEG-lipids
because it would contribute to the intermolecular interaction
by hydrogen bonding.
DPH was added to the PEG-lipid assemblies as a hydro-
phobic fluorescence probe. The emission of fluorescence
indicates the existence of a hydrophobic region in the PEG-
lipid assemblies. The packing state in the PEG50-nC16
assemblies estimated from DPH fluorescence anisotropy shows
a clear dependence of the number of acyl chains, as shown in
Figure 4. In general, the anisotropy of the bilayer membrane in
a gel state is between 0.2 and 0.3, and it decreases at a gel-to-
liquid crystalline phase transition temperature (Tc), indicating
the low packing state of the acyl chains in a liquid crystalline
Thermal Properties of the PEG-Lipids in Assembling
States. An endothermic peak at the gel-to-liquid crystalline
phase transition was observed by DSC at 64 °C (∆H ) 2.8
kcal mol-1) for PEG20-2C16 and at 54 °C (∆H ) 2 kcal
(42) Bandekar, J. Biochim. Biophys. Acta 1992, 1120, 123.
(43) Yamada, N.; Ariga, K.; Naito, M.; Matsubara, K.; Koyama, E. J.
Am. Chem. Soc. 1998, 120, 12192.

7934 J. Am. Chem. Soc., Vol. 122, No. 33, 2000
Takeoka et al.
Figure 5. The dissociation of the PEGm-nC16 from the vesicles by
dilution at 37 °C. Key: (O) PEG125-4C16; (4) PEG50-2C16; (b)
PEG125-2C16; (2) PEG50-DPPE.
Figure 4. Temperature dependence of the fluorescence anisotropy of
DPH in self-assemblies of the PEGm-nC16 amphiphiles in pure water.
Key: (O) PEG50-4C16; (b) PEG125-4C16; (3) PEG20-2C16; (4)
PEG50-2C16; (2) PEG125-2C16; (0) PEG50-1C16.
incorporated into the surface of the vesicles. This event was
detected as exothermic behavior by isothermal titration micro-
calorimetry. The exothermic rate, which would roughly represent
the incorporation rate, reached zero within 3 min, indicating
that the incorporation was in an equilibrium state. The initial
incorporation rate decreased with the increasing number of the
acyl chains of the PEG-lipids. Although the hydrophobicity
of the PEG-lipids increased with the increasing number of the
acyl chains, the probability for thermodynamically generating
enough space in the bilayer membrane to accept the PEG-
lipids should become low, which would be a determinant to
the incorporation rate. The incorporation ratios were 62, 70,
and 53% for PEG50-1C16, PEG50-2C16, and PEG50-4C16,
respectively. These ratios are reflected by equilibrium constants,
which are determined by the incorporation and dissociation rate
constants. The lower incorporation ratios of the PEG50-1C16
and PEG50-4C16 than that of PEG50-2C16 would be caused
by a larger dissociation rate constant and a smaller incorporation
rate constant, respectively.
Dissociation of the PEG-Lipids from Vesicles. Because
the PEG-lipids are incorporated into the surface of the
phospholipid vesicles in an equilibrium state, they dissociate
after diluting the vesicle dispersion. In general, PEG50 was used
for PEG-phospholipids. We have studied the dissociation
behavior of PEG50-DPPE and PEG50-DSPE from the palmi-
toyl-based vesicle (DPPC/cholesterol/DPPG at 5:5:1 molar ratio)
or stearoyl-based vesicle [1,2-distearoyl-sn-glycero-3-phosphati-
dylcholine (DSPC)/cholesterol/1,2-distearoyl-sn-glycero-3-phos-
phatidylglycerol (DSPG) at 5:5:1 molar ratio] and have con-
firmed the dissociation of PEG50-DPPE from both membranes
and the relatively slow dissociation of the PEG50-DSPE.8
We prepared vesicles, the surfaces of which were modified
with 0.2 mol % PEG chains, as a sample because the equilibrium
constant of the PEG-lipid incorporation was stable below this
incorporation ratio. After 6-fold dilution of the vesicles with a
buffer solution, the change in the incorporation ratio was
monitored as shown in Figure 5. Interestingly, PEG50-2C16
and PEG125-4C16 did not dissociate from the palmitoyl-based
vesicle but PEG125-2C16 did. Therefore, the PEG chains with
molecular weights of 5000 and 12 500 could be tethered to the
vesicular surface with two and four acyl chains, respectively.
When the PEG-lipid is compared with the PEG-phospholipid,
the former shows a higher affinity for the phospholipid bilayer
membrane. The major reason for high affinity should be the
hydrogen bonding between the ester bond of the phospholipid
and the amide bond of the PEG-lipid. Furthermore, the PEG-
phospholipid has a negative charge on the headgroup of the
phase. As shown in Figure 4, PEG50-1C16 has low anisotropy,
even at 5 °C, and decreases gradually with temperature.
PEG50-1C16 assumes a ribbonlike structure of an amorphous
liquidlike phase.
On the other hand, the PEG-lipids with two acyl chains show
a steep decrease in anisotropy around the temperature considered
Tc. These values nearly correspond to the Tc measured by DSC.
Interestingly, the molecular weight of the PEG chain of the
PEG-lipids influences the Tc; that is PEG20-2C16, PEG50-
2C16, and PEG125-2C16 have Tc values of 60, 45, and 23
°C, respectively. The lowering of Tc by increasing the molecular
weight of the PEG part would be attributed to the lower packing
of the acyl chains by steric hindrance of the PEG headgroup.
The PEG-lipid with 4C16 shows high anisotropy, at least from
10 to 90 °C, suggesting a gel state in the observed temperature
region that was independent of the molecular weight of the PEG.
Therefore, the PEG-lipid with four acyl chains has the highest
packing density. This conclusion is supported by DSC and π-A
curve measurements as already described (that is, the highest
melting point and a solid membrane with the highest collapsing
pressure). This conclusion can also be supported by the property
of a natural phospholipid, such as cardiolipin, with four acyl
chains.44
Synthetic amphiphiles with tetraacyl chains were reported by
Kunitake et al.45 They also used amino acids such as glutamic
acid or lysine as the spacers of the tetraacyl lipids and speculated
that the amphiphiles spaced with lysine were superior to
glutamic acid in orienting the acyl chains. For the design of
the hydrophobic part with several acyl chains, the orientation
of each acyl chain is the most important factor as an anchor
part so as to fit the bilayer membrane. Using a lysine dendron
structure, the orientation of all acyl chains to one side would
be possible because the lysine has enough flexibility to adjust
the direction of acyl chains24 and intramolecular hydrogen
bonding to fix those acyl chains appropriately.46
Incorporation of the PEG-Lipids into Vesicles. When the
dispersion of the PEG-lipids was mixed with a vesicle
dispersion, the PEG-lipid molecules were spontaneously
(44) Hubner, W.; Mantsch, H. H.; Kates, M. Biochim, Biophys. Acta
1991, 1066, 166.
(45) Kimizuka, N.; Ohira, H.; Tanaka, M.; Kunitake T. Chem. Lett. 1990,
1990, 29.
(46) Bosman, A. W.; Bruining, M. J.; Kooijman, H.; Spek, A. L.; Janssen,
R. A. J.; Meijer, E. W. J. Am. Chem. Soc. 1998, 120, 8547.

Synthesis and Assembly of Various Poly(ethylene glycol)-Lipids
J. Am. Chem. Soc., Vol. 122, No. 33, 2000 7935
longer PEG chain has a larger effect on preventing the
aggregation of the vesicles because the thickness of the PEG
layer covering the surface of the vesicle increases with the PEG
molecular weight. This effect would result in the suppression
of the access of macromolecules or vesicles to the surface of
the vesicle.
Conclusions
We succeeded in controlling the number of acyl chains of a
PEG-lipid by using the lysine dendron structure. The increased
number of the acyl chains resulted in the enlargement of the
hydrophobic region with a high intramolecular orientation of
the acyl chains. However, the molecular weight of the PEG
chain influenced the intermolecular packing because of steric
hindrance. The PEG-lipid with diacyl chains anchored the PEG
chain on the surface of the vesicles up to a molecular weight of
5000, whereas the PEG-lipid with tetraacyl chains anchored
the PEG chain up to a molecular weight of 12 500. Therefore,
if one would like to incorporate a longer PEG chain, one should
enlarge the hydrophobic part by increasing the generation
number of the dendron to increase the number of acyl chains.
The dispersion stability of the vesicles significantly increased
with the increasing molecular weight of the PEG chain. This
effect would lead to the prolongation of the intravenous
circulation time of the vesicles. These dendron-type hydrophobic
groups are also available to fix hydrophilic macromolecules,
such as PEG derivatives, polysaccharides, and proteins, etc, for
drug delivery systems.
Figure 6. The critical molecular weight (Mc) of the water-soluble
polymer for the aggregation of the vesicles modified with PEGm-
nC16 at 37 °C. Key: (O) plain vesicles; (b) PEG20-2C16; (4)
PEG50-DSPE; (2) PEG50-2C16; (0) PEG125-4C16; (9) PEG120-
DSPE.
phospholipid, which should lead to the low affinity of the PEG-
phospholipid for the phospholipid membrane because the surface
charge of the vesicles is negative.
Critical Molecular Weight. The solutions of PEG with
different molecular weights were added to the dispersion of the
phospholipid vesicles with 0.2 mol % PEG-lipids on the surface
for the study of the Mc for the aggregation of the vesicles. The
larger Mc indicates the higher dispersion stability of the vesicles
because of the restriction of the interaction between the vesicular
surface and the added polymers.31 The Mc of the phospholipid
vesicles (DPPC/cholesterol/DPPG ) 5/5/1 m/m) without PEG
modification is 1000, and that with PEG50-DSPE has an Mc
of 12 000. In the cases of PEG20-2C16, PEG50-2C16, and
PEG125-4C16, the Mc values are 4900, 11 700, and 49 500,
respectively (Figure 6). These results clearly indicate that the
Acknowledgment. This work was supported in part by a
project of the Material Research Laboratory for Bioscience and
Photonics in Waseda University and Health Science Research
Grants (Artificial Blood Project) from the Ministry of Health
and Welfare, Japan. Poly(ethylene glycol)s were donated by
NOF Co., Japan. We thank Kyowa Interface Science Co. for
the π-A isotherm measurements and JEOL HIGHTECH Co.
for the TEM observations.
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