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Novel Amphiphilic Ternary Polysaccharide Derivates Chitosan-g-

Novel AmphiphilicSynthesis,
PCL-b-MPEG: Ternary Polysaccharide
Characterization,Derivates Chitosan-g-
and Aggregation
PCL-b-MPEG:
in Aqueous Synthesis,
Solution Characterization, and Aggregation
in Aqueous Solution
Yiye Lu, Li Liu, Shengrong Guo
School of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, People’s Republic of China

Received 16 October 2006; revised 8 April 2007; accepted 9 April 2007


Published online 17 April 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20743

INTRODUCTION
ABSTRACT:

C
hitosan (CS) is the fully or partially deacetylated
product of chitin, which is a structural basic polysac-
Chitosan-g-PCL-b-MPEG copolymers of various
charide commonly found in nature. CS has relatively
compositions were successful synthesized via a protection- good biocompatibility and biodegradablity.1,2 Much
graft-deprotection procedure, by the esterification of attention has been paid to utilize CS in biomaterial
phthaloyl-protected chitosan (PHCS) with MPEG-b- applications such as hemostatic agent and hemodialysis
PCL-COOH, which was synthesized from MPEG and membrane, and it also has usually been prepared to micro-
or nanoparticles in drug delivery system.3,4 However, the low
e-caprolactone and carboxylated by maleic anhydride.
solubility in water, insolubility in common organic solvents
The chemical structure of the chitosan-g-PCL-b-MPEG and nonthermal plasticity of CS presents an obstacle to its
was characterized by Fourier transform infrared and application. To overcome the problem above, chemical modi-
NMR spectroscopy. The chitosan-g-PCL-b-MPEG was fication of CS should be carried out. Graft modification of
the aminopolysaccharide with synthetic polymers has been
obtained as amphoteric hybrid with amino
used as an interesting alternative method to develop novel
polysaccharide backbone and amphiphilic MPEG-b-PCL hybrid materials. Actually, most researches of grafted copoly-
side chain. Their crystallinity and aggregation behavior mers of CS are focused on nondegradable carbon chain poly-
in aqueous solution were also investigated. # 2007 Wiley mers; a few of front works have reported the synthesis of
Periodicals, Inc. Biopolymers 86: 403–408, 2007. grafted CS with degradable polyester.5–7
Polycaprolactone (PCL) is one of the presented biode-
Keywords: chitosan; polycaprolactone; poly(ethylene
gradable polyesters with excellent mechanical strength, bio-
glycol); graft copolymer compatibility and nontoxicity. It has been commonly
This article was originally published online as an accepted explored as implantable carriers for drug delivery systems
preprint. The ‘‘Published Online’’ date corresponds to the (DDS).8 Moreover, poly(ethylene glycol) (PEG) is a highly
preprint version. You can request a copy of the preprint by water-soluble polymer and frequently used for chemical
emailing the Biopolymers editorial office at biopolymers@wiley. modification of natural and artificial macromolecules for
com biomedical applications.9 In recent works, the amphipathic
block copolymer containing PEG as a hydrophilic segment
Correspondence to: Shengrong Guo; e-mail: srguo@sjtu.edu.cn and PCL as a hydrophobic one has been widely investigated
Yiye Lu and Li Liu had equal contributions to this paper.
Contract grant sponsor: Shanghai Municipal Science and Technology Commission because they can self-assemble to form micelles in aqueous
Contract grant number: 05DJ14005 media.10 It is a potential material in DDS.11,12 Nowadays, in
Contract grant sponsor: Research Start-Up Fund
the study of graft copolymers based on CS, most grafted
chains on CS have only single structure like CS-graft-poly
(methyl methacrylate) and CS-graft-PEG etc.13,14 There are
V
C 2007 Wiley Periodicals, Inc. few reports on modification of CS with such amphiphilic

Biopolymers Volume 86 / Number 5–6 403


404 Lu, Liu, and Guo

block-copolymer. Recently, Shuai et al. reported the combi- Then, the monohydroxy-terminated diblock copolymers (1
nation of activated MPEG-b-PCL-COOH with poly(ethylene mmol) and maleic anhydride (2 mmol) were dissolved in dry tolu-
ene (30 ml) and placed in a two-necked flask equipped with a mag-
imine) (PEI) for gene delivery.15 Then MPEG-b-PCL-COOH
netic stirring bar.17,18 DMAP (2 mmol) was added to the flask and
can also be conjugated on CS via esterification. The grafted the mixture under a dry nitrogen atmosphere was stirred at room
MPEG-b-PCL may endow CS with amphiphilicity. And the temperature for 48 h. Crude polymer obtained by precipitation into
DDSs based on copolymers of CS and PEG linked to readily hexane was redissolved in DCM. Then the solution was washed with
biodegradable PCL might be more desirable for in vivo appli- aqueous hydrochloric acid (10% in v/v) for three times and then
cation to enhance cell interactions and tissue permeability four times with a saturated NaCl solution. Then the organic phase
was isolated and filtered. Monocarboxy-capped diblock copolymer
due to the hydrophobic PCL moieties.
of MPEG-b-PCL-COOH recovered by precipitation into hexane
The present article is concerned with the preparation of a was vacuum-dried to a constant weight at 408C.
new type of ternary CS derivates by grafting diblock copoly-
mers of PEG and PCL. Considering the poor solubility and
Preparation of Graft Copolymers
unique structure of CS, it is important to make the reaction
First, CS was heated with excess phthalic anhydride in dried DMF to
carry out homogeneously and to maintain the aminosacchar- give phthaloylchitosan (PHCS) according to the previously reported
ide unit during the grafting process for specific properties. procedure.19,20 It was obtained as a brown-yellow powdery material
Here we connected MPEG-b-PCL-COOH with CS regioselec- and the degree of substitution (DS) of phthaloyl groups was deter-
tively at the hydroxyl group via phthaloyl-protected CS to get mined to be about 0.97 by elemental analysis. Calculated for PHCS
(DS 5 0.97) (%): C, 57.69; H, 5.26; and N, 4.91. Found (%): C,
CS-g-PCL-b-MPEG. It is expected that the amphiphilic CS-
53.90; H, 4.54; and N, 4.48.
graft copolymer forms self-aggregates in aqueous solution Then vacuum dried PHCS and monocarboxy-capped diblock
copolymers with different ratios were dissolved in DMF (PHCS 1 g
with DMF 10 ml) containing HOBt, followed by EDC  HCL added
(HOBt and EDC  HCL, each 3 moles equivalent to the diblock pre-
EXPERIMENTAL polymers).21 The reaction was continued at room temperature for
24 h under stirring. The mixture was poured into water, and the
Materials and Reagents precipitate was separated by centrifuging and was washed by DCM
CS (degree of deacetylation 5 93.34%, determined by 1H NMR to eliminate the free diblock copolymers. Then the product was vac-
spectra and element analyses, average molecular weight 5 200 K) uum-dried to a constant weight at 408C.
was purchased from JinKe Biochemical, Zhejiang, China. Two The obtained phthaloyl-protected graft copolymer (1 g) was
MPEGs (Mn 2000 and 5000) was purchased from Fluka. e-Caprolac- stirred in 10 ml of DMF and heated to 1008C under nitrogen. Hy-
tone (from Aldrich) was purified by vacuum distillation over CaH2. drazine monohydrate was added and the reaction was continued for
The 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotrizole 2 h to deprotect the phthaloyl group. The mixture was dialyzed in
(HOBt), 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydro- water and alcohol for purification and freeze-dried to obtain pow-
chloride (EDC  HCL) were from the Shanghai Yanchang biotech dery graft copolymers.
developmental, China. Phthalic anhydride, stannous(II) octoate
(SnOct), maleic anhydride, and acetic acid were all from the Rea-
gent Factory of Shanghai, China and were used as received. Characterization
All infrared spectra were obtained from samples in KBr pellets using
a Perkin Elmer Paragon 1000 FTIR spectrophotometer. 1H NMR
spectra were taken by a Varian MERCURY plus 400 at 258C. X-ray
Synthesis of MPEG-b-PCL-COOH powder diffraction diagrams were recorded with Bruker AXS X-ray
Monohydroxy-terminated diblock copolymers were first synthesized diffractometer. Elemental analyses were performed using a Perkin
by ring-opening polymerization of e-caprolactone with MPEG as a Elmer PE 2400 II el elemental analyzer.
macroinitiator and SnOct as a catalyst.16 A predetermined amount
of e-caprolactone, MPEG, and SnOct (ca. 0.1% of e-caprolactone in
molar amount) were weighed into a two-necked round-bottomed Preparation of Aggregates
flask equipped with a magnetic stirring bar. The flask was sealed The graft copolymer was dispersed in 1% acetic acid (pH 2.8) aque-
under dry nitrogen and was immersed in an oil bath at 1308C for ous solution under gentle shaking for 24 h to obtain the aggregates.
48 h. The product was purified by precipitating into hexane from All samples were prepared at room temperature and filtered using
dichloromethane (DCM) solution and was dried under vacuum at disposable 0.45-lm filters before size measurements.
358C. The mole ratio of e-caprolactone to MPEG was varied to
obtain copolymers with different PCL block length. And the degree
of polymerization of PCL block was calculated by 1H NMR spectra. Measurements of the Aggregates
The copolymer composed of MPEG (Mn 5 2000) and PCL block The effective diameters of CS self-aggregates were measured by
with molecular weight of 2000 was coded as MPEG2k-b-PCL2k. dynamic light scattering (DLS) using a Malvern Instrument Zeta-
Similarly, the block copolymers of MPEG2k-b-PCL5k and sizer Nano S laser scattering system with a 632-nm laser source. The
MPEG5k-b-PCL2k were produced, respectively. intensity of the scattered light was detected at 1738 to minimize the

Biopolymers DOI 10.1002/bip


Novel Amphiphilic Ternary Polysaccharide Derivates 405

SCHEME 1

effect of multiple scattering. Mean diameter (d) was evaluated by MPEG2k-b-PCL2k-COOH showed two resonance signals at
the following Stokes–Einstein relationship. around 6.4 ppm (doublet, C(¼ ¼O)CH¼ ¼CHC(¼ ¼O)),
kB T which are not present in the spectrum of MPEG2k-b-PCL2k
d¼ containing no maleic moiety. The result demonstrated the
3pg0 D
successful carboxylation of diblock copolymer.
where D is the diffusion constant; kB is the Boltzmann constant; T is Then graft copolymer was prepared according to the pro-
the absolute temperature; g0 is the solvent viscosity. The copolymer cedure shown in Scheme 2. In the work of Yoksan et al.,
solutions were put in 12-mm diameter polystyrene cuvettes and the PHCS has been functionalized with MPEG-COOH at the
minimum sample volume required for the experiment was 1 ml. All hydroxyl group via ester linkage in DMF.21 Similarly, the
analyses were run at 258C in six times, and the results are reported
monocarboxy-capped diblock copolymer was grafted by
as the average values.
The hydrophobic dye solubilization method with 1,6-dipheny- esterification with hydroxyl group of PHCS under EDC  HCl.
1,3,5-hexatriene (DPH, Aldrich) as a probe was used to determinate After that, phthaloyl was deprotected by incubation with hy-
the aggregates structure. A solution of CS self-aggregates which con- drazine to regenerate active amino groups in the final graft
tains 0.004 mM of DPH was placed in a sealed vial and placed in a copolymer. Figure 2 shows the FTIR spectra of CS and graft
dark place to incubate for 24 h. The concentration of sample solution
copolymer. The obtained CS-g-PCL-b-MPEG showed stron-
was varied with three levels which diluted from prepared solution.
Then, UV–vis absorption spectra of samples were recorded using a ger absorbance at 2800–3000 cm21 for CH (of CH2) and
Spectrumlab 54 UV–vis spectrometer (Lengguang Tech). The base- characteristic peaks around 1724 cm21 belonging to ester
line was from vial of corresponding polymer solution without DPH. carbonyl stretching band (C¼ ¼O), compared with the origi-
nal CS. And in Figure 3, 1H NMR spectra of graft copolymers
RESULTS AND DISCUSSION before and after deprotection (PHCS-g-PCL-b-MPEG and
CS-g-PCL-b-MPEG, repesctively), the proton signals of CS
Synthesis and Characterization of
CS-g-PCL-b-MPEGs
A successful protection-graft-deprotection route has been
reported to prepare graft copolymers of CS using PHCS as
intermediate in the previous researches.5–7 In this work, we
focused on graft conjugation of amphiphilic PCL-b-MPEGs
onto CS backbone at the hydroxyl groups while amino
groups remained free. Herein, PHCS was also introduced not
only to protect amino group but also to enable the graft reac-
tion proceed in a homogeneous system.
First of all, the diblock copolymer was prepared by ring-
opening polymerization of e-caprolactone with MPEG, and
was modified to be the corresponding intermediate prepoly-
mer (Scheme 1). 1H NMR spectra of MPEG2k-b-PCL2k and
MPEG2k-b-PCL2k-COOH were shown in Figure 1. The char-
acteristic resonances of both PCL and MPEG were detected, FIGURE 1 1H NMR spectrum of MPEG2k-PCL2k (a) and
which illustrated the synthesis of block copolymer. Moreover, MPEG2k-PCL2k-COOH (b).

Biopolymers DOI 10.1002/bip


406 Lu, Liu, and Guo

SCHEME 2

were observed at 2.8–4.5 ppm and there are characteristic sig- the terminal methyl of MPEG and the CS unit from 1H NMR
nals of both PCL and MPEG, indicating the coexistence of spectra. The signal of H-2 proton from CS is at 2.8 ppm and
two blocks in the graft copolymer. Moreover, the phthali- the signal of proton from methyl (CH3O in MPEG) is
mido peaks at 7.0–8.0 ppm became sharply weaker in Figure at 3.0 ppm. The DG could be calculated as follows:
3b. It showed that the deprotection of phthaloyl was exe-
cuted, but incompletely. All these evidence indicated the suc- I 3:0ppm
DG ¼ 3 100% ð1Þ
cessful synthesis of graft copolymers. 3 3 I 2:8ppm
On the basis of the above results, the degree of grafting
(DG) of MPEG-b-PCL branches to CS backbone in the
copolymers were determined with the signal intensities for

FIGURE 3 1H NMR spectra of PHCS-g-PCL2k-b-MPEG2k


FIGURE 2 FTIR spectra of chitosan (a) and CS-g-PCL2k-b- before deprotection in DMSO-d6 (a) and CS-g-PCL2k-b-MPEG2k
MPEG2k [DG 5 6.19] (b). [DG 5 6.19] in D2O/CF3COOD 95:5 v/v (b).

Biopolymers DOI 10.1002/bip


Novel Amphiphilic Ternary Polysaccharide Derivates 407

Table I Results of Graft Copolymerization of MPEG-b-PCL onto Chitosan

Hydrodynamic
PHCS: DGa of Diameter (nm)
Sample MPEG-b-PCL (g/g) CS-g-PCL-b-MPEG mean 6 SD (Intensity)b

CS-g-PCL2k-b-MPEG2k 1:1 1.13 202.0 6 11.1


CS-g-PCL2k-b-MPEG2k 1:3 6.19 181.3 6 12.1
CS-g-PCL2k-b-MPEG2k 1:5 6.35 162.3 6 3.2
CS-g-PCL5k-b-MPEG2k 1:1 0.09 255.7 6 8.5
CS-g-PCL5k-b-MPEG2k 1:3 1.45 223.3 6 5.5
CS-g-PCL2k-b-MPEG5k 1:1 0.44 320.0 6 6.5
CS-g-PCL2k-b-MPEG5k 1:3 3.75 299.3 6 3.8
a
Determined by 1H NMR.
b
Particles formed by the dispersal of CS-g-PCL-b-MPEG copolymers in acetate acid at a concentration of 0.5 mg/
ml. The hydrodynamic diameters of particles were measured at 258C.

where DG means the number of grafting branches per 100 stronger sharp signals appeared in the XRD pattern of CS-g-
CS units. The results of DG of different samples are shown in PCL5k-b-MPEG2k with a rather lower DG.
Table I. For graft copolymers with the fixed branch length,
like CS-g-PCL2k-b-MPEG2k, the DG increased with the
Self-Aggregation of Graft Copolymers in
increase of prepolymer amount in feed, as anticipation. But
Aqueous Solution
when its amount increased up to five times to PHCS (w/w),
When the amphiphilic CS-g-PCL-b-MPEG copolymers was
the DG was still similar to that with three times, which meant
simply dispersed in the acid water (good solvent for CS and
that the number of grafting branches maybe reached a satu-
MPEG block, but poor solvent for PCL block), they showed
rated value. The reason was probably from the stereospecific
colloidal phenomenon without a specific processing tech-
hindrance of the conjugated MPEG-b-PCL. In addition, the
nique. It is speculated that the graft copolymer self-aggregate
grafting reaction between PHCS and MPEG-b-PCL became
to form particles.
more difficult, for that with longer graft chains. The DG of
The size of self-aggregates and their distribution in acid
obtained graft copolymers was 1.45 for CS-g-PCL5k-
solution were measured by DLS. The corresponding data
MPEG2k and 3.75 for CS-g-PCL2k-MPEG5k, when MPEG-
were given in Table I. The mean diameters of aggregates are
b-PCL/PHCS ratio was 5:1 (w/w).
between 150 and 380 nm, with a unimodal size distribution.
In addition, the aggregates from graft copolymers with
higher DG had smaller size than those with low. It was also
Crystallinity
The X-ray powder diffraction patterns of CS and CS-g-PCL-
b-MPEG copolymers were detected as illustrated in Figure 4.
It was known that homopolymers of PCL and PEG were easy
to crystallize. Compared with the original CS, CS-g-PCL-b-
MPEG copolymers showed weaker and broader peaks in the
2y 5 10–258 region, indicating that the introduction of
MPEG-b-PCL branches suppressed the crystallization of CS.
When the DG increased from 1.13 to 6.19 for CS-g-PCL2k-
b-MPEG2k, there appeared two new sharp signal at 2y 5
21.48 and 2y 5 23.78 in the XRD pattern of graft copolymer,
besides the broad signal ascribed to CS backbone. This may
be attributed to the presence of crystal phase from arrange-
ment of MPEG-b-PCL side chains. The similar phenomenon
happened with the graft copolymer of CS-g-PCL5k-b- FIGURE 4 X-ray diffraction of chitosan (a), CS-g-PCL2k-b-
MPEG2k (DG 5 1.45), as shown in Figure 4d. It is probably MPEG2k [DG 5 1.13] (b), CS-g-PCL2k-b-MPEG2k [DG 5 6.19]
easier for the longer branches to form crystal phase, then (c), and CS-g-PCL5k-b-MPEG2k [DG 5 1.45] (d).

Biopolymers DOI 10.1002/bip


408 Lu, Liu, and Guo

PHCS. After deprotection, the amino group was regenerated.


The obtained CS-g-PCL-b-MPEG was amphoteric hybrid
with polyanimosaccharide backbone and amphiphilic
MPEG-b-PCL side chain. It is noteworthy that they exhibit
self-aggregates in the acid aqueous solution, which can be
applied to encapsule hydrophobic matter.

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CONCLUSION
CS-g-PCL-b-MPEG copolymers of various compositions were
successful synthesized via a protection-graft-deprotection
procedure, by the esterification of MPEG-PCL-COOH with Reviewing Editor: C. Bush

Biopolymers DOI 10.1002/bip