Preparation of a novel aggregate like sugar-ball micelle composed of poly(methylglutamate) and poly(ethyleneglycol) modified by lactose and its molecular recognition by lectin

Article abstract of DOI:10.1248/cpb.49.169

We report the preparation and characteristics of a novel micellar aggregate of an amphiphilic diblock copolymer, poly(methylglutamate) (PMG)-poly(ethyleneglycol) (PEG), whose terminus was modified by lactose lactone (LA). Due to the terminal LA moiety, th

Full text of DOI:10.1248/cpb.49.169

February 2001
Chem. Pharm. Bull. 49(2) 169—172 (2001)
169
Preparation of a Novel Aggregate Like Sugar-Ball Micelle Composed of
Poly(methylglutamate) and Poly(ethyleneglycol) Modified by Lactose
and Its Molecular Recognition by Lectin
Akiko TOYOTAMA, Shin-ichi KUGIMIYA, Junpei YAMANAKA, and Masakatsu YONESE*
Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467–8603, Japan.
Received August 24, 2000; accepted November 10, 2000
We report the preparation and characteristics of a novel micellar aggregate of an amphiphilic diblock
copolymer, poly(methylglutamate) (PMG)–poly(ethyleneglycol) (PEG), whose terminus was modified by lactose
lactone (LA). Due to the terminal LA moiety, this aggregate could be specifically recognized by RCA120 lectin.
PMG-PEG-LA was synthesized by polymerizing the N-carboxy anhydride of L-glutamic acid g-methyl ester with
H2N-PEG-LA as a polymerization initiator. By applying a fluorescence method using pyrene as a probe molecule,
we found that PMG-PEG-LA could form the aggregate in aqueous solution. Fluorescence measurements showed
that the critical aggregation concentration (C.A.C.) was 1.1؋10؊5 M. The average diameter of the aggregate was
220 nm at 25 °C, as determined by the dynamic light scattering method. Circular dichroism measurements for
the aggregate solution showed that the PMG residue took an a-helical structure, and that they associated to con-
stitute the hydrophobic core of the aggregate. By adding RCA120 lectin to the aggregate solution, the turbidity of
the solution increased rapidly, due to association of the aggregates. This implies that the aggregate could be rec-
ognized by lectin, and also suggests that sugar residues locate at the surface of the aggregates. From these find-
ings, we concluded that the PMG-PEG-LA molecules form an aggregate like a “sugar ball” micelle, whose sur-
face is covered by the sugar moieties. Application of the present aggregate system as a drug carrier is briefly dis-
cussed.
Key words peptide-based amphiphile; aggregation; recognition; sugar ball; lactose; lectin
Sugar residues on biomolecules play important roles in might be applicable as novel drug carriers.
vital phenomena in living organisms, such as differentia-
In this report, we examine the properties of novel peptide-
tion, senescence and tumorigenic transformation.1) In these based amphiphiles such as the formation of the aggregate
processes, the sugar moieties operate as key components for like “sugar-ball” micelles, and their molecular recognition
molecular recognition. Thus, sugars have considerable signif- due to the sugar residue.5—7) We synthesized an amphiphilic
icance for living systems, not merely as energy sources. It is diblock copolymer composed of poly (ethyleneglycol), PEG,
important to clarify how the sugar residue performs its bio- and poly(methylglutamate), PMG, whose ethyleneglycol ter-
function as a host in living systems, and in what cases it rec- minal was modified by lactose lactone, LA, (hereafter desig-
ognizes guest molecules. Substantial studies have been made nated as PMG-PEG-LA). Unmodified PMG-PEG block
to elucidate the recognition mechanism for sugars, by con- copolymer was also synthesized for comparison. The PMG-
structing artificial host molecules,2,3) and furthermore, host PEG-LA aggregate was recognized by RCA120 lectin which
supermolecules such as dendrimers, both of which have has a specific affinity for lactose.
sugar residues.4)
Host supermolecules also have practical significance, in Experimental
Materials The modified block copolymer PMG-PEG-LA was obtained
as shown in Fig. 1 by polymerizing the N-carboxy anhydride of L-glutamic
acid g-methyl ester in dimethylformamide (DMF) with H2N-PEG-[O-b-
galactopyranosyl-(1→4)]-D-gluconamide (H2N-PEG-LA) as a polymeriza-
terms of applications as a drug delivery system (DDS). One
can solubilize hydrophobic drugs in the hydrophobic core of
the dendrimer, which is available as a novel drug carrier.4)
tion initiator.8) H2N-PEG-LA was synthesized from lactose lactone and H2N-
PEG-NH2 (Degree of polymerization of PEGϭ75 and 113, purchased from
Sigma Co., Ltd.). Lactose lactone was synthesized from lactose hydride
(purchased from Sigma Co., Ltd.), and the presence of the carbonyl group
was confirmed using IR spectroscopy. The IR spectrum was obtained with a
1600 series FT-IR (Perkin-Elmer Inc., CT, U.S.A.). The N-carboxy anhydride
of L-glutamic acid g-methylester was synthesized from L-glutamic acid g-
methylester (purchased from Sigma Co., Ltd.) using triphosgene (purchased
from Nakalai Tesque Co., Ltd. Osaka, Japan) in tetrahydrofuran (THF). The
molar ratio of the anhydride to the initiator was chosen to be 30. The poly-
merization reaction was carried out at room temperature for 72 h. The de-
grees of polymerization for the PMG segment (DPPMG) in the copolymer
were determined by 1H-NMR method using a Lambda 400 spectrometer
(JEOL, Co., Ltd., Tokyo) in trifluoroacetic acid (TFA) with tetramethyl
silane (TMS) as an internal standard. The peak areas of b- and g-methylene
protons and methylene protons were compared to those for PMG (chemical
shift qϭ2.0—2.8 ppm and 3.9—4.1 ppm, respectively). The degrees of poly-
merization for the PEG segment (DPPEG) were equal to those of H2N-PEG-
However, because of their small sizes, the amount of the
drugs included in the dendrimers is restricted. On the other
hand, peptide-based amphiphilic polymers have been re-
ported to form micelles with ordered cores consisting of pep-
tide residues having a-helical structures. If the micelle sur-
face is covered with sugar residues, it is expected to take a
“sugar ball” like structure. These micellar systems have vari-
ous advantages as drug carriers: 1) The structure is com-
pletely reversible depending on the amphiphile concentra-
tion, namely, whether it is above or below a critical micelle
concentration. 2) The size of the micelle can be controlled by
varying the length of the peptide and/or the hydrophilic parts
and it is much larger than that obtainable by using den-
drimers. Therefore, the peptide-based micellar systems can
solubilize larger amounts of drugs than in the case of den-
drimer systems. Thus, the “sugar ball” micellar systems NH2. DPPMG and DPPEG values were confirmed by means of TOF-Mass spec-
To whom correspondence should be addressed. e-mail: yonese@phar.nagoya-cu.ac.jp © 2001 Pharmaceutical Society of Japan

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Vol. 49, No. 2
Fig. 1. Synthesis of PMG-PEG-LA
Table 1. Characteristics of PMG-PEG-LA and PMG-PEG
DPPEG
75
DPPMG
32
C.A.C./M
PMG-PEG-LA
1.1ϫ10Ϫ5
PMG-PEG 1
PMG-PEG 2
113
113
50
20
7.7ϫ10Ϫ7
1.4ϫ10Ϫ6
Fig. 2. Synthesis of PMG-PEG
troscopy. TOF-Mass measurements were performed using a Voyager-Elite
type mass spectrometer (PerSeptive Biosystem Inc., CA, U.S.A.).
The unmodified amphiphilic polymer, PMG-PEG, was obtained as shown
in Fig. 2 by polymerization of the N-carboxy anhydride of L-glutamic acid g-
methylester in DMF with w-amino poly(ethyleneglycol) methylester as an
initiator. Two kinds of PMG-PEG block copolymers having different seg-
ment lengths were synthesized (hereafter designated as PMG-PEG 1 and 2).
The values of DPPEG and DPPMG were obtained in the same manner as for
PMG-PEG-LA. DPPEG and DPPMG values for these samples are compiled in
Table 1.
Determination of Critical Aggregation Concentration (C.A.C.) The
critical aggregation concentrations (C.A.C.) of the amphiphilic copolymers
in aqueous solutions were determined by using pyrene as a fluorescent
probe.10—13) Fluorescence intensity of the polymer solutions was measured
in the presence of pyrene at various polymer concentrations, C, at a wave-
length of 393 nm. The value of C.A.C. was estimated from the change of flu-
orescence intensity with C. The measurements were performed by using a
fluorescence spectrophotometer (Type FP-77, JASCO Co., Tokyo, Japan) at
room temperature.
Characterizations of the Aggregate and the Core Structures of PMG
Radii of the aggregates were determined by a dynamic laser light scattering
apparatus (DLS-700, Otsuka Electron Co. Ltd., Tokyo, Japan). Sample solu-
tions were carefully filtered before every measurement to eliminate dusts.
The secondary structures of the polypeptide PMG in the cores of the aggre-
gates were studied by applying circular dichroism (CD) spectroscopy using a
JASCO J-720 CD spectrometer.
Recognition of the PMG-PEG-LA Aggregate by Lectin The recogni-
tion of the aggregates was investigated by adding lectin, RCA120, (Sigma
Co., Ltd.) to the solutions of PMG-PEG-LA and PMG-PEG using a turbid-
ity method. The turbidity t was measured by UV-VIS spectrophotometer
(Type U-3000, Hitachi Co. Ltd., Tokyo, Japan) at a wavelength of 500 nm.
The polymer concentrations were 50 times larger than the C.A.C. values and
they formed aggregates in the sample solutions. In order to confirm the
binding between RCA120 and aggregates of PMG-PEG-LA or PMG-PEG,
free lactose was added to samples containing the aggregates 34 h after
preparations, and the accompanying change of t values measured.
Fig. 3. Relative Fluorescent Intensity, I/I0, vs. Amphiphilic Concentration,
C, Plot for Aqueous Solutions of PMG-PEG-LA at 25 °C, Measured at
Wavelengthϭ392.6 nm
this concentration it increased rapidly. Hence, the C.A.C.
value for PMG-PEG-LA was 1.1ϫ10Ϫ5 M. A similar trend is
commonly observed for micellar systems. The increase of
I/I0 above a critical micellar concentration is attributed to an
increase of micelle number (not micelle size) with C.14)
Similar measurements for PMG-PEG 1 (DPPMGϭ50) and 2
(DPPMGϭ20) showed that their C.A.C. values were 7.7ϫ10Ϫ7
and 1.36ϫ10Ϫ6 M.15) The decrease of C.A.C. with increasing
DPPMG appears to be due to an increase in hydrophobicity.
The higher C.A.C. value for PMG-PEG-LA compared to that
for PMG-PEG 1 and 2 was attributable to an increase in hy-
drophilicity due to the sugar residues.
Structure of the Aggregate CD spectra of PMG-PEG-
LA and PMG-PEG were measured at C’s above their
Results and Discussion
C.A.C. of the Peptide-Based Amphiphiles The critical C.A.C.’s. Figure 4 demonstrates the molar ellipticity [q]l of
aggregation concentrations (C.A.C.) of amphiphilic polymers PMG-PEG-LA and unmodified PMG-PEG in aqueous solu-
were determined by fluorescence spectroscopy as described tions. C values were 2.24ϫ10Ϫ4 M and 3.88ϫ10Ϫ5 M, respec-
above. Figure 3 shows the dependence of the relative fluores- tively. The spectra for both amphiphiles showed a sharp max-
cence intensity (I/I0) on the polymer concentrations, C, for imum at lϭca. 190 nm followed by a shallow single mini-
aqueous solutions of PMG-PEG-LA. I is the fluorescence mum at lϭca. 230 nm. Generally, the CD spectrum of an a-
intensity of the sample, and I0 is the intensity of the solvent. helical structure for an isolated peptide shows a peak at
I/I0 were almost constant below Cϭ1.1ϫ10Ϫ5 M, while above lϭ190 nm and two minima at lϭ208 and 222 nm. On the

February 2001
171
Fig. 4. Circular Dichroism Spectra of PMG-PEG-LA and PMG-PEG in
Aqueous Solution
Fig. 5. Change of Turbidity, t, with Time, t for Aqueous Solutions of
PMG-PEG-LA (A, B) and PMG-PEG (C), by Addition of RCA120 and Lac-
tose
[PMG-PEG-LA]ϭ2.24ϫ10Ϫ4
M
and [PMG-PEG]ϭ3.88ϫ10Ϫ5 M. (——): PMG-
PEG-LA, (------): PMG-PEG.
RCA120 and lactose were added at tϭ0 and tϭ34 h, respectively. [PMG-PEG-
LA]ϭ1.12ϫ10Ϫ4, [PMG-PEG]ϭ7.76ϫ10Ϫ6. [Lactose]ϭ3.04 mg (A) and 30.4 mg (B,
C), t was measured at wavelength of 500 nm and 25 °C.
other hand, when the helixes are associated, the peak wave-
length shifts toward higher l, and the first minimum is ab-
sent.17) The present CD profiles are attributable to associated
a-helixes, which presumably form the hydrophobic core of
an aggregate particle.
We note that the sizes of the aggregates were comparable
with the wavelengths of the incident light for the CD mea-
surements, as will be mentioned below. In this case, diffused
scattering of the incident beam by the aggregates might af-
fect the CD spectra.18) Although this can not readily be cor-
rected, we believe that it does not affect the profiles quantita-
tively, since a similar profile for associated helical polypep-
tides has often successfully been measured for micellar sys-
tems.19)
Fig. 6. Schematic Representation of Micelle of PMG-PEG-LA Formed in
Aqueous Solution
The present results indicate that PMG segments in both
PMG-PEG-LA and PMG-PEG block copolymers take an a-
helical structure, and that they associated to form the hy-
Recognition of the PMG-PEG-LA Aggregate by Lectin
drophobic cores of the aggregates. Furthermore, the observa- The interaction between the aggregate of PMG-PEG-LA and
tion that the presence of a LA residue does not largely affect RCA120 lectin molecules in aqueous solutions was evaluated
the helix structure strongly suggests that the LA moiety is by measuring the change of turbidity, t, at a wavelength of
located on the surface of the PMG-PEG-LA aggregates. 500 nm. By adding RCA120 lectin to PMG-PEG-LA solutions
This is reasonable in light of the hydrophobic nature of the at C ϾC.A.C., the value of t increased after about 1 h as
PMG segments and the hydrophilic properties of the LA shown in Fig 5. This is presumably caused by association of
residue.
the aggregates, since the hydrophilicity of the aggregates is
The average diameter d of the aggregates were determined largely reduced by binding of the lectin. In contrast, for the
by applying the DLS method. The d values for PMG-PEG- aggregate of PMG-PEG 1, the change of t value was negligi-
LA and unmodified PMG-PEG were 220 and 270 nm, ble on addition of the lectin. These results indicated that the
respectively. Measurements were performed at Cϭ2.24ϫ lactose residues of the PMG-PEG-LA aggregate are located
10Ϫ4 M and 3.88ϫ10Ϫ5 M, which were chosen to be much on the surface of the aggregate, and are successfully recog-
higher than the C.A.C. values, but sufficiently dilute to avoid nized by RCA120 lectin.
interparticle hydrodynamic effects.20) The particle size distri-
Furthermore, in order to examine the reversibility of bind-
bution was relatively narrow. For example, the standard devi- ing between RCA120 and the sugar residues, free lactose was
ation in the particle size for PMG-PEG-LA aggregate was added to the solution of the complexes 34 h after preparation
approximately 50%, as analyzed by the cumulant method.21) (Fig. 5). Two experiments were performed so that lactose
This relatively uniform particle size distribution suggests that concentrations in the final solutions were 19.3 and 3.2 mM.
the resulting aggregate has a micelle-like structure. This is By adding lactose, t values rapidly decreased. The drop of t
consistent with the findings from the CD measurements. The was greater with the larger amount of lactose. However, t
somewhat smaller d value for the modified amphiphile can values decreased only by about 25—30% and did not recover
be explained in terms of augmented hydrophilicity by the LA the initial value. On the other hand, the t value for PMG-
the residue, which reduces the association number in the ag- PEG did not significantly change on addition of lactose as
gregates as seen in the micellar system.
shown in Fig. 5.

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Vol. 49, No. 2
References and Notes
From these results, we conclude that RCA120 lectin binds
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free lactose is needed to remove lectin from the complexes,
due to the competing substitution reaction with free lactose.
A possible model of the PMG-PEG-LA aggregate is
shown in Fig. 6. It is reasonable to assume that PMG-PEG-
LA molecules are able to form aggregate like “sugar ball”
micelles with the sugar moiety on the surface and with a
rigid hydrophobic core of a-helixes of PMG.
The present aggregate could be applicable as a novel drug
carrier. One of the advantages of utilizing the PMG-PEG-LA
system is that its size can be controlled easily by changing
DPPMG and DPPEG. We note that the size of the present aggre-
gate (dϭ220 nm) should be reduced for future use as a drug
carrier. It is usually desirable to design the aggregate size to
be smaller than 100 nm, i.e. virus size. Thus, further studies
on shorter amphiphiles are required, however, aggregates
with moderately large size should enable confinement of
larger amounts of hydrophobic drugs than sugar-ball den-
drimers.4) Furthermore, the specific recognition ability of
PMG-PEG-LA aggregate like sugar-ball micelles demon-
strated above appears to be potentially useful for designed
drug targeting.22)
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16)
1.36ϫ10Ϫ6
M
and higher than that of the present sample. The de-
crease of C.A.C. can be explained in terms of reduction of hydropho-
bicity with DPPMG
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Conclusions
PMG-PEG-LA block copolymers form aggregate like
“sugar ball” micelles, with the sugar moiety on the outside of
the spherical micelle. The aggregate diameter determined by
the dynamic light scattering method was approximately
200 nm. The aggregate formed a complex with RCA120
lectin, including the presence of recognition sites on the sur-
face that recognized lectin specifically. These findings sug-
gest the possibility of application of the present system as a
novel drug carrier.
Acknowledgments This work was supported by the Sasakawa Scientific
22) Hashida M., Takakura Y., J. Controlled Release, 31, 163—171 (1994).
Research Grant from The Japan Science Society.