New amphiphilic biodegradable β-cyclodextrin/poly(l-leucine) copolymers: Synthesis, characterization, and micellization

Article abstract of DOI:10.1016/j.carbpol.2010.01.003

A new amphiphilic biodegradable β-cyclodextrin/poly(l-leucine) (β-CD-PLLA) copolymer was synthesized by ring-opening polymerization of N-carboxy-l-alanine anhydride (LL-NCA) in N,N-dimethylformamide (DMF) initiated by mono-6-amino-β-cyclodextrin (H₂N-β-CD). The structures of the copolymers were determined by IR, ¹H NMR and GPC. The fluorescence technique was used to determine the critical micelle concentrations (CMC) of copolymer micelle solution. The diameter and distribution of micelles were characterized by dynamic light scattering (DLS) and its shape was observed by transmission electron microscopy (TEM). The results showed that LL-NCA could be initiated by H₂N-β-CD to produce the copolymer. These copolymers could self-assemble into nano-micelles in water. The CMC of copolymer solution and the size of micelle reduced with increasing proportion of the hydrophobic part. TEM images demonstrated the micelles are all spherical. Such block copolymers could be expected to find applications in drug delivery systems and other biomedical fields.

Full text of DOI:10.1016/j.carbpol.2010.01.003

Carbohydrate Polymers 80 (2010) 885–890
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
New amphiphilic biodegradable b-cyclodextrin/poly(L-leucine) copolymers:
Synthesis, characterization, and micellization
*
Guolin Zhang, Fang Liang, Ximing Song, Daliang Liu, Mengjiang Li, Qiuhua Wu
Key Laboratory of Green Synthesis and Preparative Chemistry of Advanced Materials, The Education Department of Liaoning Province, College of Chemistry,
Liaoning University, Shenyang 110036, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 June 2009
Received in revised form 12 December 2009
Accepted 4 January 2010
Available online 7 January 2010
A new amphiphilic biodegradable b-cyclodextrin/poly(L-leucine) (b-CD–PLLA) copolymer was synthe-
sized by ring-opening polymerization of N-carboxy-L-alanine anhydride (LL-NCA) in N,N-dimethylform-
amide (DMF) initiated by mono-6-amino-b-cyclodextrin (H2N-b-CD). The structures of the copolymers
were determined by IR, 1H NMR and GPC. The fluorescence technique was used to determine the critical
micelle concentrations (CMC) of copolymer micelle solution. The diameter and distribution of micelles
were characterized by dynamic light scattering (DLS) and its shape was observed by transmission elec-
tron microscopy (TEM). The results showed that LL-NCA could be initiated by H2N-b-CD to produce
the copolymer. These copolymers could self-assemble into nano-micelles in water. The CMC of copolymer
solution and the size of micelle reduced with increasing proportion of the hydrophobic part. TEM images
demonstrated the micelles are all spherical. Such block copolymers could be expected to find applications
in drug delivery systems and other biomedical fields.
Keywords:
b-Cyclodextrin
Poly(L-leucine)
Amphiphilic biodegradable copolymers
Micelles
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
able rate of these poly(a-amino acid)s, hydrophilic functional
groups may be introduced into the polymer chain. Poly(ethylene
glycol) (PEG) was usually introduced into the copolymer as hydro-
philic segment to enhance the copolymer’s hydrophilicity because
of its excellent hydrophilicity and biocompatibility (Lavasanifar,
Samuel, & Kwon, 2002). There are many reports about copolymers
of amino acids which were synthesized from PEG so as to regulate
the hydrophilicity and biodegradable rate, such as poly(aspartic
Amphiphilic block and graft copolymers can self-assemble into
micelles. The micelles have unique characteristics such as nano-
size, a core–shell architecture, and a good thermodynamic stability
in physiological condition because of their low critical micelle con-
centration (CMC) (Tian et al., 2005). The micelles formed by amphi-
philic biodegradable block and graft polymers may be used for
controlled release of drug implants, proteins, medical devices,
etc. because they possesses both biodegradability and amphiphilic-
ity (Bikram et al., 2004; Soppimath, Aminabhavi, Kulkarmi, & Rud-
zinski, 2001).
acid)/poly(ethylene glycol) block copolymer, poly(
tamate)/poly(ethylene glycol) block copolymer, poly(
poly(ethylene glycol) block copolymer, poly( -alanine)/poly(ethyl-
c
-benzyl
L-glu-
L
-lysine)/
L
ene glycol) block copolymer and so on (Harada & Kataoka, 1995,
1998; Kugo, Ohji, Uno, & Nishino, 1987; Zhang, Ma, Li, & Wang,
2003). These amphiphilic copolymers could be self-assembled into
nanoscaled micelles in suitable medium and some have been used
as carriers of drug delivery systems (Kataoka et al., 2000; Li &
Kwon, 2000). However, there are few reports about the completely
biodegradable amphiphilic copolymers which were synthesized
from hydrophobic poly(amino acid)s with hydrophilic and biode-
gradable products.
Poly(a-amino acid)s (PAA) are well known as very important
synthetic biodegradable materials. Due to their polypeptide back-
bone, they have the potential to be degraded in biological environ-
ments. Moreover, since they have low immunogenicity, good
biocompatibility and excellent mechanical properties, they may
be widely used in pharmaceutical and other medical fields (Fukuo-
ka, Uyama, & Kobayashi, 2004; Iwata, Matsuda, Mitsuhashi, Itoh, &
ˇ
Ikada, 1998; Rypácek, 1998). But it is well known that most com-
monly used PAAs, such as poly( -leucine), poly( -alanine),
poly( -benzyl glutamate), etc. are rather hydrophobic and degrade
a
a
Cyclodextrins (CDs) are a family of cyclic oligosaccharides that
c
are composed of
duced from starch by enzymatic degradation. The most common
CDs are of three types: -cyclodextrin ( -CD), b-cyclodextrin (b-
CD) and -cyclodextrin ( -CD) (Girek, Kozlowski, Koziol, Walk-
a-1,4-linked glucopyranose subunits. CDs are pro-
very slowly by simple hydrolysis under human body conditions. In
order to improve the hydrophilicity and to control the biodegrad-
a
a
c
c
owiak, & Korus, 2005). Among them, b-CD which contains seven
glucopyranose units is the most accessible, the lowest-priced and
* Corresponding author.
E-mail address: qiuhuawu@yahoo.cn (Q. Wu).
0144-8617/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.01.003

886
G. Zhang et al. / Carbohydrate Polymers 80 (2010) 885–890
generally the most useful. It has low toxicity, hydrophilicity, excel-
lent biocompatibility and biodegradability (Gao, Wang, Fan, & Ma,
2005; Prabaharan & Mano, 2006). One of the most remarkable
applications of b-CD is its use as drug carriers in controlled release
systems. As drug carriers, b-CD allows the solubilization, stabiliza-
tion, and transport of hydrophobic drugs together with several
pharmacological benefits such as the reduction of unwanted side
2.4. Synthesis of b-cyclodextrin/poly(L-leucine) (b-CD–PLLA)
copolymer
Certain amounts of H2N-b-CD and LL-NCA were dissolved to-
gether in anhydrous N,N-dimethylformamide (DMF) and kept at
25 °C for 72 h in nitrogen atmosphere under stirring. The reaction
mixture was poured to 200 ml of distilled water to give white pre-
cipitate. The precipitate was filtered off and washed with distilled
water and THF, respectively, then dried in vacuum at room temper-
ature for 24 h to give the desired b-CD–PLLA copolymers. The reac-
tion scheme is shown in Scheme 1. Different molar ratios of the
feeding NCA to H2N-b-CD resulted in the corresponding copoly-
mers with various compositions as listed in Table 1.
effects (Prabaharan & Gong, 2008). Poly(L-leucine) (PLLA) is one
of the synthetic biodegradable polypeptides, if the PLLA chain is
combined with b-CD to prepare an amphiphilic completely biode-
gradable polymers, its hydrophilicity and biodegradability can be
regulated, and thus its applications may extend widely.
In this work, a new amphiphilic completely biodegradable
b-cyclodextrin/poly(
L-leucine) (b-CD–PLLA) copolymer was syn-
thesized by ring-opening polymerization of N-carboxy-
L
-leucine
2.5. Characterization of the copolymers
anhydride (LL-NCA) using mono-6-amino-b-cyclodextrin (H2N-b-
CD) as a initiator. Their structures were characterized and some
properties of the copolymers were also investigated.
The IR spectra were collected by a Perkin-Elmer FT-IR spectrom-
eter using KBr disks. Elemental analysis was performed on Thermo
Electron Flash EA 1112 instrument. 1H NMR spectra were mea-
sured on a Varian Mercury-300 NMR spectrometer at room tem-
perature, using CDCl3 as solvent. Chemical shifts (d) were given
in ppm using tetramethylsilane (TMS) as an internal reference.
The gel permeation chromatography (GPC) measurement was con-
ducted with a Waters 1515 GPC instrument equipped with a HT 3
column (effective molecular-weight range: 500–30,000) and a
2414 differential refractive index detector. THF was used as eluent
at the flow rate of 1.0 ml/min at 30 °C, and the molecular weights
were calibrated with polystyrene standards.
2. Experimental
2.1. Materials
L-Leucine (biochemical reagent) was purchased from Sinop-
harm Chemical Reagent Co. Ltd. (Shanghai, China) and dried in vac-
uum for 24 h before use. Triphosgene (chemical reagent) was
obtained from Haining Zhonglian Chemical Reagent Co. Ltd. (Zhe-
jiang, China), used without any treatment. b-Cyclodextrin (b-CD,
biochemical reagent, >98%) was purchased from Aoboxing Biotech
Company Ltd. (Beijing, China), used without any treatment. p-Tol-
uenesulfonyl chloride (TsCl, chemical reagent) was purchased from
Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) and recrys-
tallized in the mixed solvents of chloroform and n-hexane before
use. Tetrahydrofuran (THF, analytical reagent) was purchased from
Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), dried and
distilled in the presence of sodium immediately before use. N,N-
dimethylformamide (DMF, analytical reagent) was supplied by Sin-
opharm Chemical Reagent Co. Ltd. (Shanghai, China), and dried
over CaH2 and distilled before use. Other chemicals are all analyt-
ical reagents made in China and used without further purification.
2.6. Preparation of micelles
The micelles were prepared using a solvent displacement meth-
od with a tetrahydrofuran/water (THF/H2O) system (Wilhelm et al.,
1991). A copolymer (50 mg) was first dissolved in 5 ml of THF in a
100 ml round bottomed flask, and then 40 ml of doubly distilled
water was slowly added into the copolymer solution. The THF
was removed using rotary evaporator at 25 °C for 2 h. The solution
was transferred into a 50 ml volumetric flask after dilution to the
calibration line with doubly distilled water to get 1 g/l micelles.
2.7. Micelle characterization
The critical micelle concentrations (CMC) of the copolymers
were measured by fluorescence technique using pyrene as a probe.
Pyrene solution (0.1 mg/l in acetone) was added into a series of
volumetric flasks in such an amount that the final concentration
of pyrene in each sample solution was 2.47 Â 10À7 mol/l after dilu-
tion to the calibration line, and then the acetone was removed
completely. The polymer solution and doubly distilled water were
added into the volumetric flasks containing the pyrene to obtain
desired copolymer concentrations from 5 Â 10À5 to 1.0 mg/ml.
The samples were sonicated for 15 min, heated at 50 °C for 2 h,
and cooled to room temperature for overnight to equilibrate the
pyrene and micelles. Steady-state fluorescence spectra were ob-
tained on a Varian Cary Eclipse fluorescence spectrophotometer.
For emission spectra, kex = 339 nm, and for excitation spectra,
kem = 390 nm. The scan rate was 120 nm/min and the slit opening
was 2.5 nm. The size distribution of micelles was determined by
dynamic light scattering (DLS) using Malvern Nano ZS instrument
at 25 °C. The morphology of the micelles was investigated by trans-
mission electron microscopy (TEM), carried out on a JEM-100SX
electron microscope, operating at an accelerating voltage of
80 kV. Specimens were prepared by dipping copper grids into
aqueous solutions of copolymers. The grids were then left to stand
on a piece of filter paper and air dried before measurements.
2.2. Preparation of mono-6-amino-b-cyclodextrin (H2N-b-CD)
H2N-b-CD was prepared according to a literature procedure
(Matsumoto, Noguchi, & Yoshida, 1998). First, b-CD and p-toluene-
sulfonyl chloride (TsCl) reacted to obtain mono-6-(p-tolylsulfo-
nyl)-b-cyclodextrin (TsO-b-CD). Anal. calcd. for C49H76O37S (%): C:
45.65, H: 5.94, O: 45.92. Found (%): C: 45.78, H: 5.75, O: 45.82.
Then, TsO-b-CD reacted with sodium azide (NaN3) to give mono-
6-azido-b-cyclodextrin (N3-b-CD). Anal. calcd. for C42H69N3O34
(%): C: 43.49, H: 5.99, N: 3.62. Found (%): C: 43.76, H: 6.02, N:
3.57. Finally N3-b-CD reacted with ammonium hydroxide
(NH3ÁH2O) to give mono-6-amino-b-cyclodextrin (H2N-b-CD).
Anal. calcd. for C42H71NO34 (%): C: 44.48, H: 6.31, N: 1.24. Found
(%): C: 44.62, H: 6.54, N: 1.21. The reaction route is shown in
Scheme 1.
2.3. Preparation of N-carboxy-L-leucine anhydride (LL-NCA)
N-carboxy-L-leucine anhydride (LL-NCA) was prepared by the
reaction of -leucine with triphosgene in dried THF at 50 °C accord-
L
ing to a literature procedure (Daly & Poché, 1988), melting point
76–77 °C. Yield: 80%. The reaction route is shown in Scheme 1.

G. Zhang et al. / Carbohydrate Polymers 80 (2010) 885–890
(OH)14
(OH)14
887
(OH)14
(OH)14
TsCl, pyridine
40°C
Ph3P, NH 3.H2O
DMF, 25°C
NaN3
H2O, 80°C
(
)
7
(
)
6
(
)
(
)
6
OH
HO
O
OTs
HO
N3
HO
NH2
6
-CD
TsO- -CD
N3- -CD
H2N- -CD
(OH)14
O
O
O
CH3
C
C
NH2
CH3
CH3
Cl3COCOCCl3
H2N- -CD
DMF, 25°C
CH2CHCH3
H
CH3CHCH2
CH3CHCH2
CH NH
CH COOH
O
THF, 50°C
HO
NH C C
N
H
n
(
)
6
H
L-leucine
LL-NCA
-CD-PLLA
Scheme 1. The route for the synthesis of b-cyclodextrin/poly(
L
-leucine) copolymer.
Table 1
Related data on b-CD–PLLA copolymers.
H2N-b-CD/NCAa
Wb-CD/WPLLA
Yield (%)
Mn
Mn
Mw/Mn
CMC (g/l)
Micelle size (nm)d
b
b
c
Sample
b-CD–PLLA10
b-CD–PLLA30
b-CD–PLLA50
1/10
1/30
1/50
53/47
27/73
17/83
44.6
50.0
45.3
2150
4185
6558
2079
3986
6159
1.26
1.25
1.21
2.63 Â 10À3
1.77 Â 10À3
1.23 Â 10À3
201
168
142
a
b
c
Molar ratio of H2N-b-CD to NCA in feed.
Determined by 1H NMR in CDCl3 solution.
Determined by GPC in THF at 30 °C.
d
Determined by dynamic light scattering in aqueous solution at 25 °C.
observed at 1655 and 1554 cmÀ1, respectively, indicating the for-
mation of the polypeptide block. No carbonyl absorptions of NCA
at 1820 and 1758 cmÀ1 appeared in the spectra of all products,
which indicated that no NCA residue co-existed in the polymer
samples.
3. Results and discussion
3.1. Synthesis of b-cyclodextrin/poly(L-leucine) (b-CD–PLLA)
copolymer
The 1H NMR spectra of b-CD–PLLA were shown in Fig. 2. The
peaks at 0.82 and 0.93 ppm were assigned to protons of the –CH3
units of the PLLA segment. The peaks at 0.98–1.82 ppm were as-
signed to protons of the b-CD. The peak at 2.65 ppm was assigned
to protons of the –CH2 units of the PLLA segment. The peaks at
2.88–2.96 ppm were assigned to protons of –OH group in the b-
CD. The peak at 4.21 ppm was assigned to protons of the –CH
group in the –COCHNH– units of the PLLA segment. 8.03 ppm
was assigned to proton of N–H group in the copolymer.
The GPC traces of the copolymers were shown in Fig. 3. The
three copolymers showed unimodal molecular weight distribution.
This further indicated that the copolymerization was completed
successfully and there was no homo-polymer in the reaction prod-
uct. GPC data of the copolymers are listed in Table 1.
It is well known that primary amines, being more nucleophilic
than basic, can be used as initiators for the ring-opening polymer-
ization of NCA to prepare poly(a-amino acid)s, undergoing a nucle-
ophilic addition to the carbonyl group of the NCA (Gotsche, Keul, &
Höcker, 1995). Because H2N-b-CD contains primary amine group, it
can initiate ring-opening polymerization of LL-NCA to form copoly-
mer. A series of the copolymers with various molecular weights
were synthesized and the results were summarized in Table 1. It
was found that the total molecular weights of the copolymers in-
creased with the molar ratio of the feeding monomer NCA to the
initiator.
The IR spectra of b-CD–PLLA copolymers were shown in Fig. 1.
The absorption peak at 3297 cmÀ1 was assigned to mNH stretch
vibration, and the typical amido absorption bands I and II were
CD-PLLA1
CD-PLLA2
CD-PLLA3
(OH)14
CH3
CH2CHCH3
O
H
HO
NH
C
C
N
H
n
(
)
6
H
CDCl3
4000 350 0 3000 2500 2000 1500 1000
Wavenumber(cm-1)
500
3
ppm
9
8
7
6
2
1
4
5
Fig. 1. IR spectra of b-CD–PLLA copolymers.
Fig. 2. 1H NMR spectrum of b-CD–PLLA10 copolymer in CDCl3.

888
G. Zhang et al. / Carbohydrate Polymers 80 (2010) 885–890
observed with increasing concentration of b-CD–PLLA10, indicat-
2.5
2.0
1.5
1.0
0.5
0.0
a β-CD-PLLA10
b β-CD-PLLA30
c β-CD-PLLA50
ing that micellization takes place for the b-CD–PLLA copolymer.
Such results can be attributed to the transfer of pyrene molecules
from water to a hydrophobic environment within the micelle core.
The onset of micellization and the critical micelle concentra-
tions (CMC) can also be obtained from the studies of excitation
spectra (Deng et al., 2007). For the copolymer b-CD–PLLA10, 334
and 339 nm are chosen as the peak wavelength of the (0, 0) band
in the pyrene excitation spectra in the aqueous phase and in the
entirely hydrophobic core of polymeric micelle, respectively. The
pyrene fluorescence intensity ratios (I339/I334) are plotted against
the logarithm of copolymer concentration. The plots are shown
in Fig. 5. Below a certain concentration, I339/I334 is constant, above
this concentration, I339/I334 increases with increasing lgC and final-
ly reaches a plateau. From this plot, the critical micelle concentra-
tion (CMC) of 2.63 Â 10À3 g/l was obtained from the intersection of
two straight lines: the base line and the rapidly rising I339/I334 line.
The CMC of b-CD–PLLA30 and b-CD–PLLA50 were also obtained
from the same methods and listed in Table 1. The CMC values were
in the magnitude of 10À3 g/l and reduced with the increasing of the
proportion of hydrophobic parts, indicative of the amphiphilic nat-
ure of b-CD–PLLA copolymers.
c
a
b
4.5
4.0
3.5
logMw
3.0
2.5
Fig. 3. GPC traces of b-CD–PLLA copolymers.
1
0.5
70
0.1
0.05
0.02
0.01
0.005
0.002
0.0005
0.0002
0.0001
0.00005
(g/L)
60
50
40
30
20
10
0
3.3. Size and size distribution of b-CD–PLLA micelles
The size and size distribution of micelles were measured by
DLS. As shown in Fig. 6, the mean diameter of micelles formed
by b-CD–PLLA10, b-CD–PLLA30, and b-CD–PLLA50 were about
201, 168 and 142 nm, respectively. The size of micelle was reduced
with the increasing of the proportion of hydrophobic part, so the
size of the copolymer micelle could be adjusted by changed the
proportion of hydrophobic part of the copolymer. The PLLA chain
in the copolymer was a poly(
a-amino acid) like protein. Therefore,
it would form a -helix by intramolecular hydrogen bonding or a
a
b-sheet by intermolecular hydrogen bonding in aqueous solution.
When PLLA content increases, the intermolecular hydrogen bond-
ing is enhanced. The micellar aggregation number increases, then
the density increases and the particle size decreases (Liu, Zhu,
Zhao, Jiang, & Wu, 2000).
300
310
320
330
340
350
360
Wavelength(nm)
Fig. 4. Excitation spectra of pyrene as a function of b-CD–PLLA10 concentration in
water.
3.2. Formation of micelles
3.4. Morphology of b-CD–PLLA micelles
Pyrene has been widely used as a probe to monitor the associ-
ation and micellization of macromolecules in solutions because its
photophysical character changes with the variation of the existing
environment (Li, Lin, Chen, & Zhang, 2006). The micellar structures
of b-CD–PLLA are confirmed by fluorescence technique using pyr-
ene as a probe. The fluorescence excitation spectra of pyrene of
1.98 Â 10À7 mol/l in the presence of b-CD–PLLA10 at various con-
centrations are shown in Fig. 4. A red shift from 334 to 339 nm is
The morphology of the micelles was examined by TEM. Fig. 7
presents the TEM images of the micelles. They were all spherical.
The micelle size formed by b-CD–PLLA10, b-CD–PLLA30, and b-
CD–PLLA50 were about 160, 120 and 90 nm, respectively. The
diameter data were smaller than that determined by DLS, because
the micelle diameter determined by DLS represents their hydrody-
namics diameter while that obtained by TEM is related to the col-
lapsed micelles after water evaporation.
2.0
c
a
b
2.0
1.5
1.0
0.5
1.5
1.0
0.5
1.5
1.0
0.5
-1
0
-5
-4
-3
-2
-1
-5
-4
-3
-2
lgC
-1
0
-5
-4
-3
-2
lgC
lgC
Fig. 5. Plots of I339/I334 versus logarithm of b-CD–PLLA10 (a), b-CD–PLLA30 (b) and b-CD–PLLA50 (c) concentrations.

G. Zhang et al. / Carbohydrate Polymers 80 (2010) 885–890
889
12
c
a
10
8
b
12
10
8
10
8
6
6
6
4
4
4
2
2
2
0
0
0
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
Diameter(nm)
Diameter(nm)
Diameter(nm)
Fig. 6. The size distributions of b-CD–PLLA10 (a), b-CD–PLLA30 (b) and b-CD–PLLA50 (c) copolymer micelles in water measured by DLS.
Fig. 7. TEM images of b-CD–PLLA10 (a), b-CD–PLLA30 (b) and b-CD–PLLA50 (c) copolymer micelles.
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L-lactide)s as
In this study, a new amphiphilic completely biodegradable b-
CD–PLLA copolymer was synthesized by ring-opening polymeriza-
tion of N-carboxy-L-leucine anhydride (LL-NCA) using mono-6-
L
a
a
amino-b-cyclodextrin (H2N-b-CD) as a initiator. Characterizations
using IR, 1H NMR and GPC had confirmed the designed structure.
It could self-assemble into nano-micelles in water. The CMC value
measured by fluorescence spectroscopy was in the magnitude of
10À3 g/l and decreased with the increasing of the proportion of
hydrophobic parts. The average micelle size determined by DLS re-
duced with the increasing of the proportion of hydrophobic parts.
TEM images demonstrated that they were all spherical. This
amphiphilic property would be useful in drug delivery systems
and other biomedical fields.
&
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Acknowledgement
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This work was supported by the Natural Science Foundation for
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051, 2009A306).
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