Synthesis and characterization of graft copolymer of chitosan

and polyethylene glycol

Yan Zhao

Yiyi Sun

Zhihe Zang

Xiaohong Xu

Zhonglin Zhang

Ling Zhong

Wang Zan

Yan Zhao

Lin Sun
Received: 22 March 2010 / Accepted: 8 November 2010 / Published online: 23 November 2010
Springer Science+Business Media B.V. 2010
Abstract MPEG was modied with 1,1
0
-carbonyldiim-
idazole, then the activated MPEG reacted with primary
amino groups of chitosan. Synthesize the graft copolymer
of chitosan and polyethylene glycol in two steps. The
structure of the copolymer was characterized by FT-IR and
1H-NMR. It agrees with the PEG content of classical
stealth nanoparticles materials. The X-ray diffraction and
DSC analysis proved that the crystallinity of the copolymer
increased. It is a promising material for the stealth nano-
particles. It is a potential new carrier for the drug delivery
systems of long-circulation and solid carcinoma.
Keywords Chitosan MPEG Graft copolymer
Stealth nanoparticles Carrier material
Introduction
The stealthy non-liver targeting long-circulating nano-
particles has became one of the research focuses in drug
delivery systems [1]. The synthesis and application of
covalently linked hydrophilic polymers as the carrier for
stealthy nanoparticles has drawn much attention [28]. In
the 1990s of the 20th Century, the self-assembling supra-
molecular complexes were widely researched as a novel
carrier for stealthy nanoparticles [4, 5]. The carrier was
devised according to the self-assembling phenomenon of
amphoteric polymers and the hydrophilic surface of the
novel carrier has long limp chains. Those amphoteric
polymers assembly automatically in water and form a
dimensionally stable structure with hydrophilic crust and
hydrophobic core. Those micelle nanoparticles formed by
the self-assembly of polymers have hydrophilic surfaces
with long limp chains [913]. The structure of the micelle
nanoparticles make them difcult to be absorbed by the
opsonin in plasma, and helps them to evade the identi-
cation and absorption of reticuloendothelial system [9,
1416]. So it is easier for this micelle nanoparticles to
achieve long circulation [17]. The micelle nanoparticles
have low toxicity, good bio-compatibility, and promoted
permeability to physiological barriers and the vessel walls
of tumor tissues [5, 1827]. Chitosan is a positive charged
natural alkaline polysaccharide with good bio-compatibil-
ity and moderate tumor afnity. The self-assembling
polymer micelle nanoparticles made of the copolymer of
chitosan and PEG has positive charged hydrophilic surface
and long limp chains, and it is an ideal carrier for stealthy
nanoparticles [5, 1827].
The aim of the present work was to synthesize the
promising material for the stealth nanoparticles. With CS
(MW 200000, de-acetyl rate [99%) and MPEG5000 as the
materials, MPEG was activated by 1,1
0
carbonyldiimidaz-
ole. This reaction was gentle and usually was used in
protein-PEG. The activated MPEG was made with acti-
vation rate of 109% by choosing the best solvent, ratio of
materials and reaction time, and the solubility or activation
rate and recovery rate were used as the guidelines. Then,
the activated MPEG reacted with primary amino groups of
chitosan and the graft copolymer of chitosanMPEG was
obtained through two steps by choosing the best solvent,
reaction temperature, reaction time and ratio of materials
and with the graft rate, determined by gravimetric method
Y. Zhao Y. Sun Z. Zang X. Xu Z. Zhang L. Zhong
W. Zan Y. Zhao
Chengdu Medical College, Chengdu 610083, Sichuan, China
L. Sun (&)
West China Hospital, Sichuan University,
Chengdu 610041, Sichuan, China
e-mail: linsun@live.cn
1 3
Mol Biol Rep (2011) 38:24552462
DOI 10.1007/s11033-010-0381-y
and indirect method, as the guidelines. It could be as a
potential new carrier for the drug delivery systems of long-
circulation and solid carcinoma.
Materials and methods
Reagents and instruments
Chitosan (CS, viscosity-average molecular weight of
200000 D, Degree of deacetylation [99%; Zhejiang Yuh-
uan Ocean Biochemical Co, Ltd.); single-methoxy Poly-
ethylene glycol 5000 (MPEG5000, MPEG), AR, Alfa Aesar;
l,l-carbonyldiimidazole (carbonyldiimidazole, CDM; Zhe-
jiang Linhai Kaile Chemical Factory); other drugs or
reagents were AR reagents.
85-2 type temperature magnetic stirrer (Shanghai Sile
Instruments Division); UV-2201 spectrophotometer (Shi-
madzu, Japan); Fourier Transform infrared spectrometer,
(NICOLET, United States); Elemental Analyzer (CAR-
LOERBA, Italia); Unity INOVA-400/54 high-resolution
ultra-NMR (Varian; United States); X-ray powder dif-
fraction Multifunction Instrument (Philips, Netherlands);
differential scanning calorimetry (NETZSCH, German).
Synthesis of a activated single-methoxy
polyethylene glycol 5000
MPEG was activated by 1,1
0
-carbonyldiimidazole. This
reaction was gentle and usually was used in protein-PEG.
With reference to Ohko and Deshpande methods [28, 29],
with some slight modications. The suitable amount of
MPEG and the CDM were dissolved in dioxanedimethyl
sulfoxide (2:1), which was mixed steadily with 18 h. After
it was cooled to room temperature, the precipitated product
of anhydrous diethyl ether was added with the ice water
bath. Then ltering, washing with cold dioxanedimethyl
sulfoxideanhydrous diethyl ether (2:1:3) for two times,
and then washing with cold anhydrous ether for three
times. Vacuum drying for the reserve. MPEG activation
reaction is as follows:
Characterization of the activated single-methoxy
polyethylene glycol 5000
Synthetic compounds were characterized by elemental
analysis and FT-IR to conrm the structure.
Synthesis of the chitosanPEG graft copolymer
The suitable amount of CS with the MPEGCDM was dis-
solved in 0.7% HCI solution, which was mixed steadily for
18 h at 37C. After cooling to room temperature and drop-
ping with 0.5 mol/l NaOH to pH 78, the precipitated
product of acetoneanhydrous diethyl ether (1:2) was added
with the ice water bath. The ice water bath was added
dropwise under stirring, an anhydrous ether (1:2) precipita-
tion product. Then ltering with G4 glass sand funnel, and
washing with cold water:acetone (1:10). Vacuum drying
at 80C for the reserve and x the weight. Synthesis of
CSMPEG reaction equation is:
Determine the structure and graft rate of the CSPEG
The structure of the copolymer was identied with FT-IR
and
1
H-NMR and graft rate of the copolymer was deter-
mined to be 11% by gravimetric method, indirect method
CH3-(O-CH2-CH2)n-OH +
N
N C
O
N
N
N
N
O
C CH3-(O-CH2-CH2)n-O-
O- (CH2-CH2-O)n-CH3
CH3 (CH2-CH2-O)n- O-
O
C
C
O
n
) (
H
H
CH2-O
NH
H
OH
H
H
O
O
O
O
H
H
OH
H
NH2
CH2-OH
H
H
( )
n
N
N
O
C CH3-(O-CH2-CH2)n-O- +
or
O
O
H
H
OH
H
NH
CH2-OH
H
H
( )
n
O
C O- (CH2-CH2-O)n-CH3
2456 Mol Biol Rep (2011) 38:24552462
1 3
and
1
H-NMR. X-ray diffraction and DSC tested the char-
acterization of the copolymer.
Results and discussion
The structural characterization of MPEGCDM
In the present study, the activation of MPEG with CDM
was the rst-step reaction to make the PEG of amino acids
or proteins. The mechanism of this response was clear, and
the structure of reaction product was also determined.
Therefore, only IR and elemental analysis were used to
analyze and determine its structure.
Element analysis
MPEGCDM was performed for C, H, N elemental anal-
ysis, the results was shown in Table 1. The activation
degree of MPEG was indicated with the relative value of
measured contents and theoretical content of the element-
N. Batches of MPEGCDM were synthesized, and the
activation degrees were about 104%.
Infrared spectroscopy analysis
After the FT-IR detection, the reaction product of t
OH
about 3400 cm
-1
was disappeared, and the reaction prod-
uct of t
C=O
about 1762 cm
-1
was noticeable. The results
showed that MPEGCDM was activated completely
(Fig. 1). By the result of elemental analysis and FT-IR it
showed that the synthesized compound in the step was the
target compound.
The reaction was switched to dioxanedimethyl sulf-
oxide (2:1) as the solvent with much higher degree of
activation when compared with the more reported literature
with dioxane or acetone as the solvent [28, 29]. Analyzing
the reasons, it may be because of the good solvent of the
compound solvent reactant, and the reaction is more
complete. Meanwhile, some of the PEG may be mixed with
the raw materials of MPEG, so that the measured degree of
activation was better than 100%.
Infrared spectroscopy analysis
The FT-IR spectrum of Cs and CSMPEG were shown in
Fig. 2, 1590 cm
-1
was a NH2 plane bending vibration
peak of CS, and NH2 absorption peak in CSMPEG was
signicantly decreased. All of this indicated that reaction
mainly occured in NH2 of CS. Meanwhile, primary and
secondary alcohol absorption peak within the scope of
10301200 cm
-1
of CS were no signicantly weakened or
disappeared, which indicated that C6 and C3 was not
occurred the esterication.
1
H-NMR of CS, MPEG, and CSMPEG
1H-NMR spectrum of CS, MPEG, and CSMPEG were
shown in Fig. 3. Due to the interference of solvent, only the
resonance absorption peak of d
3.88
(3H, CH), d
3.69
(5H,
CH), and d
3.14
(2H, CH) of CS with lessly and easily to
distinguish when compared with the standard CS spectrum.
The resonance absorption peak of d
3.51
(CH
3
), d
3.45
(CH
2
),
and d
3.37
(CH
2
) of MPEG could be easy seen. The reso-
nance absorption peak of d
3.48
(MPEGCH
2
), d
3.41
(MPEG
CH
2
), and d
3.19
(2H, CSCH) of CSMPEG was also easy
to distinguish, especially in d
3.41
(MPEGCH
2
) was sepa-
rated well. Therefore, the ratio of d
3.19
(2H, CSCH) and
d
3.41
(MPEGCH
2
) peak area could be used to calculate the
graft rate of CSMPEG:
graft rate
A
d
3:41
=2
A
d
3:19

2:19=2
10:00
100% 10:95%
From the results of FT-IR and 1H-NMR, it could be con-
rmed that the synthesis of CSMPEG was the target
compounds.
X-ray diffraction analysis
The X-ray diffraction diagrams of CS, MPEG, MPEG
CDM, and the CSMPEG were shown in Fig. 4. The
spectra of MPEG and MPEGCDE are basically the same,
but with the CS and CSMPEG were greatly different: the
diffraction peaks were shown for CSMPEG in 2h (angle
of 31.7 and 56.5, respectively), which were not existed
for the CS and MPEG, and the crystal spacing d was
0.28 nm (relative abundance 23.3%) and 0.16 (relative
abundance 3.7%) respectively. At the same time, the rel-
ative abundance of the diffraction peaks for CSMPEG in
the 45.2 was increased from 1.85 to 14.3%. All of the
above indicated that due to the introduction of MPEG for
the synthesis of new compounds, and the formation of a
new intramolecular hydrogen bonds, so that the crystalli-
zation of chitosan molecules and the ordered structure of
the original were changed, crystallinity were increased, and
solubility were decreased, indicating that the material CS is
Table 1 Elementary analysis of MPEGCDM
Element Theoretical value (%) Observed value (%)
C 54.36 54.42
H 9.04 9.17
N 0.55 0.57
Mol Biol Rep (2011) 38:24552462 2457
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more conducive to co-precipitation methods such as prep-
aration of NP [30, 31].
DSC analysis
CS, MPEG, physical mixture of CS and MPEG, and
CSMPEG for differential scanning calorimetry analysis,
the results were shown in Fig. 5. It can be seen from the
diagram, for the physical mixture of CS and MPEG, the
peak location and peak area of 100.9C endothermic peak
(peak area 173.8 J/g) of CS and 63.1C endothermic
peak (peak area 151.0 J/g) of MPEG did not change. For
the CSMPEG, the location and peak area of 100.9C
endothermic peak (peak area 173.6 J/g) of CS was
unchanged, but 63.1C endothermic peak of MPEG was
moved to the left of 57.2C (peak area 243.6 J/g).
According to the grafting rate of 10.95%, the theoretical
peak area of the endothermic peak of CS and MPEG was
156.8 J/g with much smaller than the peak area of CS and
MPEG in the CSMPEG (417.2 J/g). The results showed
that the degree of crystallinity for the CSMPEG was much
higher than the physical mixture of CS and MPEG, which
was consistent with the results of X-ray diffraction.
Synthesis of CSMPEG and the grafting rate
The testing methods of PEGNH2 with a compound in the
former literature of were through the trinitrobenzene sul-
fonic acid (TNBS), uorescamine, gel permeation chro-
matography (GPC), capillary zone electrophoresis (CE),
Fig. 1 FT-IR spectrum of
MPEG (a) and MPEGCDM (b)
2458 Mol Biol Rep (2011) 38:24552462
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matrix-assisted laser desorption mass spectrometry
(MALDI-MS), Raman spectra, Fourier transform infrared
spectroscopy (FT-IR), and so on. As the limitations of
methods and equipment, etc., the most commonly used
method of measurement is mainly the TNBS method. This
method is an indirect method by determining the reduction
in the number of NH2 to calculate the degree of activation
or modication, but NH2 in the solution have been con-
cealed or TNBS was inaccessible to the sites of free amino,
so the determination of the accuracy of this method is poor.
Meanwhile, the accuracy of TNBS method was also
affected by the interference of polyethylene glycol, and
must separate out the free polyethylene glycol to
re-determinate it. In addition, this method requires a
quantitative reaction under the alkaline conditions. If the
reactants were not dissolved in the solution of alkaline
(e.g., CS), the determination is meaningless. With refer-
ence to the former literatures [32], by using two-phase
system spectrophotometric to determinate the concentra-
tion of free polyethylene glycol, then calculating the
grafting rate in an indirect method. After the inspection, the
determined results of CS, CDM, and CSMPEG were not
interfered. After several batches of samples were mea-
sured, the results of indirect method, the weight of law and
the
1
HNMR measurement are consistent with each other
with both 1011%. Therefore, the two methods are suitable
methods for the determination of the grafting rate of
polymer.
Fig. 2 FT-IR spectrum of CS
(a) and CSMPEG (b)
Mol Biol Rep (2011) 38:24552462 2459
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Conclusion
In conclusion, with CS (MW 200000, de-acetyl rate [99%)
and MPEG5000 as the materials, MPEG was activated by
1,1
0
carbonyldiimidazole. This reaction was gentle and
usually was used in protein-PEG. The activated MPEG was
made with activation rate of 109% by choosing the best
solvent, ratio of materials and reaction time, and the sol-
ubility or activation rate and recovery rate were used as the
guidelines. Then, the activated MPEG reacted with primary
amino groups of chitosan and the graft copolymer of
chitosanMPEG was obtained through two steps by
choosing the best solvent, reaction temperature, reaction
time and ratio of materials and with the graft rate, deter-
mined by gravimetric method and indirect method, as the
guidelines. The structure of the copolymer was identied
with FT-IR,
1
H-NMR and graft rate of the copolymer was
determine to be 11% by gravimetric method, indirect
method and
1
H-NMR. The graft rate agreed with the PEG
content of classical stealth nanoparticles materials. X-ray
Fig. 3 1H-NMR spectrum of
CS (a), MPEG (b), and
CSMPEG (c)
2460 Mol Biol Rep (2011) 38:24552462
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diffraction and DSC proved that the crystallinity of the
copolymer raised and the copolymer had a tendency to
form self-assembling micelle [913]. The copolymer was
stable at room temperature and was a promising carrier for
stealthy micelle nanoparticles [5, 1827]. It was not
reported at home and overseas that the graft copolymer of
CSMPEG, with the PEG content of classical stealth
nanoparticles materials, was synthesized through two steps.
The synthesis of CSMPEG graft copolymer is expected to
become the carrier of stealth nanoparticles, and it will be
benet for long-circulating drug delivery systems, and
provide the solid foundation for tumor research in the
future.
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