Photo-responsive shell cross-linked micelles based on carboxymethyl chitosan and their application in controlled release of pesticide

Article abstract of DOI:10.1016/j.carbpol.2015.06.077

An amphiphilic carboxymethyl chitosan conjugate with photolabile 2-nitrobenzyl side groups (NBS-CMCS) was synthesized, which could self-assemble into polymeric micelles in aqueous condition following by adding dropwise dialdehyde to form a cross-linking s

Full text of DOI:10.1016/j.carbpol.2015.06.077

Carbohydrate Polymers 132 (2015) 520–528
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Photo-responsive shell cross-linked micelles based on carboxymethyl
chitosan and their application in controlled release of pesticide
Zhao Ye, Jingjing Guo, Dewei Wu, Mingyuan Tan, Xiong Xiong, Yihua Yin∗, Guanghua He∗
Department of Pharmaceutical Engineering, School of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, Wuhan 430070,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
An amphiphilic carboxymethyl chitosan conjugate with photolabile 2-nitrobenzyl side groups
(NBS–CMCS) was synthesized, which could self-assemble into polymeric micelles in aqueous condition
following by adding dropwise dialdehyde to form a cross-linking structure. TEM and 1H NMR confirmed
that the cross-linked micelles had a core–shell configuration with an average diameter of 140 5.5 nm.
DLS and TEM observations showed that the cross-linked micelles were stable in aqueous solution at pH
7.0 without light irradiation, while they could transfer into nanocapsules upon exposure to 365 nm UV
light. Diuron, a photosynthetic inhibitor, was encapsulated in the cross-linked micelles reaching encap-
sulation efficiency as much as 91.9%. No release of the encapsulated diuron was detected without light,
whereas a release rate as high as 96.8% over 8 h at pH 7.0 buffer was observed under solar stimulated
irradiation, indicating that the cross-linked micelles may be used as a photo-controlled sustained release
carrier for the delivery of photosynthetic inhibitor.
Received 21 March 2015
Received in revised form 16 June 2015
Accepted 17 June 2015
Available online 2 July 2015
Keywords:
Carboxymethyl chitosan
Core–shell micelles
Photo-responsibility
Pesticide delivery
© 2015 Elsevier Ltd. All rights reserved.
1. Introduction
2006; Feng et al., 2013) because their hydrophobic core can serve
as a reservoir for various hydrophobic bioactive agents; whereas
The wide use of pesticide in agriculture contributes to achiev-
ing global food security but brings undesirable effects to humans
and the natural environment. As much as 90% of the applied con-
ventional pesticides never reach their objects to produce desired
biological response at the precise time and in precise quantities
required, due to nonspecific and periodic application of active
agents. This not only increases the cost but also leads to environ-
mental pollution (Margni, Rossier, Crettaz, & Jolliet, 2002; Yi et al.,
2011). Because of the cost and other limitations in the synthesis
of new pesticides, increasing attention is being paid to the devel-
opment of controlled delivery systems of the pesticides (Sun et al.,
2014; Tasmin et al., 2014; Yi et al., 2011). Controlled delivery tech-
nology offers the opportunity to develop formulations which can
improve the performance of pesticides by increasing their efficacy
and safety and making them environmentally less harmful.
the outer shell consisting of hydrophilic block of polymer confers
aqueous solubility and steric stability, avoiding the inactivation of
loaded bioactive molecules in delivery process. Importantly, PMs
can resist the dilution in environmental fluid due to lower criti-
cal micelle concentration (CMC) as compared to surfactant-based
micelles. Recently, PMs with photoresponsive property have gained
considerable interest (Jiang, Tong, Morris, & Zhao, 2006), because of
their ability to exert spatial and temporal control over the release
of active substance in respond to external light stimuli. Generally,
the construction of photoresponsive PMs need use a phototrig-
ger such as coumarin, 2-nitrobenzyl, 7-nitroindoline (He, Tong,
& Zhao, 2009; Jiang, Qi, Lepage, & Zhao, 2007; Wang, Ma, Wang,
& Zhang, 2007) or their derivatives. Among them, 2-nitrobenzyl
has attracted particular attention since it can undergo a photolysis
reaction under UV light or near-infrared light (NIR), breaking the
hydrophilic–hydrophobic balance of the 2-nitrobenzyl-containing
polymer and leading to the disruption of PMs. For example,
the micelle system based on an amphiphilic chitosan conjugate
containing 2-nitrobenzyl groups was successfully synthesized by
assembly technique, which could release hydrophobic anticancer
drug camptothecin in response to UV-light and showed a better
cytotoxicity against MCF-7 cancer cells (Meng et al., 2013). So far,
Polymeric micelles (PMs) have been increasingly investigated
as promising controlled delivery carriers for bioactive substances
(Li et al., 2012; Mathot, van Beijsterveldt, Preat, Brewster, & Arien,
∗
Corresponding authors. Tel.: +86 027 87859300; fax: +86 027 87859300.
E-mail addresses: yihuayin@aliyun.com (Y. Yin), guanghuahe@126.com (G. He).
http://dx.doi.org/10.1016/j.carbpol.2015.06.077
0144-8617/© 2015 Elsevier Ltd. All rights reserved.

Z. Ye et al. / Carbohydrate Polymers 132 (2015) 520–528
521
however, the photo-responsive PMs or nanospheres reported in
the literature are mostly used as a carrier for controlled release of
drugs, and the designs for encapsulation of pesticides for control-
ling release have not been reported.
titrated by NaOH solution (0.1 mol/L). The amount of base at the
inflection point was calculated by second-order derivative method.
The DS of carboxymethyl groups was obtained according to the for-
mula: DS = Mcts [(V2–V1) CNaOH/m]/[1–58 (V2–V1) CNaOH/m], m is
the mass of titrated carboxymethyl chitosan. Mcts is the molar mass
ofthe glucosamineunits;V1 is the titration end ofexcesshydrochlo-
ric acid; V2 is the titration end of carboxyl. The obtained total DS
of carboxymethyl groups was about 0.75. The substitution degree
of N-carboxymethyl group was about 0.22. The viscosity-average
Herbicides are a group of pesticides that control or eliminate the
growth of weeds. Herbicides account for more than 40% of the pesti-
cides consumed globally each year, and are the most important and
broadly used agrochemicals in rice culture systems (Yamamoto &
Nakamura, 2003). Herbicides have many different modes of action,
one of which is inhibiting photosynthesis, a process vital to weed
survival (Sopen˜a, Maqueda, & Morillo, 2009). When the process
is disrupted for any reason, the weeds will die. Photosynthesis,
therefore, is the target of a group of herbicides known as photosyn-
thetic inhibitors. Photosynthesis requires light energy from the sun.
When weeds are exposed to sunlight if a photo-controlled delivery
system arrived at the target can release photosynthetic inhibitor,
photosynthesis will be inhibited.
molecular weight (M␩) of CMCS was calculated according to clas-
1.0
sic Mark–Houwink equation [␩] = 7.92 × 10−5 × M␩
(Luo, Teng,
Wang, & Wang, 2013) by measuring the intrinsic viscosity [␩] of
CMCS aqueous solution using an ubbelohde viscometer, its value
was 5.15 × 105 Da.
2.3. Synthesis of 2-nitrobenzyl succinate (NBS)
In this work, we attempted to synthesize a novel amphiphilic
carboxymethyl chitosan conjugate (NBS–CMCS) by side-chain
grafting of hydrophobic photosensitive 2-nitrobenzyl succinate
(NBS) to the main chains of CMCS, and then using the conjugate
to develop a photo-responsive shell cross-linked nanocarrier for
delivery and controlled-release of photosynthetic inhibitor or her-
bicide. Diuron is chosen as a hydrophobic photosynthetic inhibitor
which is encapsulated in the nanocarrier. The encapsulation stabil-
ity and photo-triggered release behavior were evaluated. It was
expected that this preparation did not release the encapsulated
diruon or only a little amount was released without light irra-
diation, while it could release out the inhibitor to inhibit the
photosynthesis of weeds once exposed to sunlight.
Typically, 2-nitrobenzyl alcohol (3.83 g, 25.0 mmol), succinic
anhydride (5.03 g, 50.0 mmol), and DMAP (1.53 g, 12.5 mmol) were
dissolved completely in CHCl3 (86 mL). The reaction mixture was
refluxed overnight (24 h) under a nitrogen atmosphere. CHCl3 was
evaporated under reduced pressure. The mixture was washed three
times with 10% HCl and then extracted with saturated NaHCO3
solution. The basic aqueous phase was washed with diethyl ether
and acidified to pH 5.0 with 10% HCl. The white solid precipitate
was collected and dried in vacuo at 40 ◦C overnight to provide the
product (NBS) (5.63 g, 89%). All above reactions were in dark place.
2.4. Synthesis of 2-nitrobenzyl succinate-carboxymethyl chitosan
(NBS–CMCS)
NBS–CMCS was synthesized by grafting NBS onto the main
chain of CMCS according to the method in literature (Kindermann,
George, Johnsson, & Johnsson, 2003). In briefly, CMCS (110 mg)
was dissolved completely in 15 mL deionized water. NBS
(25.8 mg, 0.1 mmol), EDC (28.8 mg, 0.15 mmol), and NHS (17.3 mg,
0.15 mmol) were added into DMSO (3.0 mL) in sequence, and
the reaction mixture was stirred at ambient temperature for 6 h.
After that, the reaction mixture was dropwise added into the
CMCS solution, and the final reaction mixture was stirred at ambi-
ent temperature for 24 h. The resulting mixture was dialyzed in
deionized water for two days and followed by lyophilization to
produce NBS–CMCS. The product was characterized by 1H NMR
spectroscopy and FT-IR spectroscopy.
In the various feed molar ratios of amino groups of CMCS and
carboxyl groups of NBS, three samples were synthesized using
the same method and named as NBS–CMCS-1, NBS–CMCS-2, and
NBS–CMCS-3, respectively. The DS of NBS was determined by mea-
suring the concentration of unreacted NBS in the reaction mixture
through UV absorbance at 260 nm and calculated according to
2. Materials and methods
2.1. Materials
Chitosan (CS, Mw = 5.6 × 105 Da) (99%) with a deacetylation
degree of 91.13% was purchased from the Zhejiang Yuhuan
Biotechnology Company. Succinic anhydride, O-nitrobenzyl alco-
hol, 4-(dimethylamino) pyridine (DMAP), N-hydroxysuccinimide
(NHS), glutaraldehyde (GA) (25% in water), and Nile red were pur-
chased from Acros Organics and used without further purification.
1-Ethyl-3-(3-(dimethylamino) propyl) carbodiimide hydrochlo-
ride (EDC) (≥99%) was obtained from Titan Chemical Corp. Dialysis
tube (MWCO, 14 and 3.5 kDa) was obtained from Shanghai Lvniao
Technology Corp. All the other chemicals were purchased as
analytical-reagent grade from Sinopharm Chemical Reagent Corp.,
and used as received.
2.2. Synthesis of carboxymethyl chitosan (CMCS)
the equation: DS = (Mtotal − Munreacted)/Mglucosamine. Where Mtotal
,
CMCS was synthesized according to the literature (Yin, Lv,
Tu, Xu, & Zheng, 2010). In briefly, 10 g of chitosan was stirred
with isopropanol (100 mL) for 6 h and then filtered. NaOH solu-
tion (50%, wt%) was added to the beaker with the filter residue,
and this solution was fully stirred to mix evenly and then was
frozen overnight. Chloroacetic acid (28.0 g, 0.29 mol) dissolved in
isopropanol (30.0 mL) was then dropped onto the frozen chitosan
mixture, and the mixture was stirred at a low temperature (10 ◦C)
for 20 h. This solution was then filtered and the filter residue
was washed with absolute ethanol. The crude product was then
removed by centrifugation and subsequent dialysis against deion-
ized water, and then freeze–dried to obtain CMCS. The degree
of substitution (DS) of carboxymethyl groups was determined by
potentiometric titration (Muzzarelli, Tanfani, Emanuel, & Mariotti,
1982). Briefly, CMCS (0.3 g) dissolved in HCl (50 mL, 0.1 mol/L) was
M
unreacted, and Mglucosamine are the amount (in moles) of NBS in
feed, NBS unreacted in reaction mixture, and glucosamine unit
in CMCS (Meng et al., 2013). The equation for calibration curve
is A = 0.0093C + 0.0008, R2 = 0.9981, A is the absorbance of NBS at
260 nm UV light, C is the concentration of the NBS. The DS of NBS on
CMCS in three samples were 0.035, 0.066 and 0.081, respectively.
2.5. Preparation of micelles
NBS–CMCS (10.0 mg) was dissolved completely in 10 mL deion-
ized water and pH value of the solution was adjusted to pH 7.0
under mild stirring, followed by sonication for 3 min using a probe
type sonifier (JY92-2D, made by Ningbo Xinzhi Bio-tech Co., Ltd.)
at 100 W using a pulse function (pulse on 2.0 s, pulse off 2.0 s), the
micelle solution was obtained. The solution was filtrated through

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Z. Ye et al. / Carbohydrate Polymers 132 (2015) 520–528
Table 1
The mean diameters and distributions of uncross-linked micelles and cross-linked micelles based on NBS–CMCS.
Conjugates
Uncross-linked micelles
Particle diameter (nm)
Cross-linked micelles
Particle diameter (nm)
Polydispersity index
Zeta potential (mV)
Polydispersity index
Zeta potential (mV)
NBS–CMCS-1
NBS–CMCS-2
NBS–CMCS-1a
NBS–CMCS-2b
242 0.04
226 0.15
253 0.12
40.6 5.13
0.097 0.012
0.104 0.016
0.114 0.010
6.136 0.012
−29.2 1.42
−25.5 1.24
−28.2 1.30
−30.5 1.11
217 0.02
196 0.11
220 0.03
238 0.20
0.082 0.008
0.074 0.012
0.072 0.010
0.065 0.014
−29.6 1.11
−26.2 1.03
−29.4 1.01
−29.2 1.12
a
The data from the NBS–CMCS-1 micelles stored for one month in the dark at pH 7.0.
The data from the NBS–CMCS-2 micelles exposed to UV radiation for 10 min at pH 7.0.
b
0.45 ␮m Millipore filter and the filtrate was divided into two parts.
One part was directly used for the preparation of shell cross-linked
micelles, the other was dialyzed against pH 7.0 solution, and then
used for analysis and testing. All operations were carried out at the
dark environment.
Diuron dissolved in 50% aqueous acetone was added dropwise into
the solution and stirred for 10 h. Then the whole solution was trans-
ferred into a dialysis bag and dialyzed against deionized water for
12 h. Water was changed once every 2 h after 4 h. The dialyzed
aqueous solution was collected for diuron content determination
in uncross-linked micelles.
Using the same method as in Section 2.5, the diuron-loaded
cross-linked micelles was prepared by adding dropwise of GA aque-
ous solution (1.35 × 10−2 mmol/mL) to the above diuron-loaded
uncross-linked micelle solution, and then used for diuron content
determination.
To determine diuron loading efficiency (LE) and encapsulation
efficiency (EE), 10 mL of diuron-loaded micelles dispersion was
centrifugated at high speed of 10,000 rpm for 20 min, washed three
times with a little bit of deionized water to remove the free diuron.
The resultant precipitate was freeze–dried for release study. The
concentrations of diuron in the decanted aqueous solution and
three washing solutions were determined by HPLC using a C18
bonded reverse phase column with a mobile phase of acetonitrile-
water mix (80:20, v/v), at a flow rate of 1.0 mL/min. The peak was
detected at 250 nm with a UV detector. The LE and EE were calcu-
lated as follows:
The preparation of shell cross-linked micelles was carried out as
follows: 1 mL of GA aqueous solution (1.35 × 10−2 mmol/mL) was
added dropwise into the above micelle solution over 4 h under vig-
orous stirring at 25 ◦C. The resulting mixture was kept stirring for
another 6 h and then further purified by dialysis (MWCO 3500 Da)
in PBS solution for 24 h, and used for analysis and testing.
The degree of crosslinking (DC) of GA was estimated by mea-
suring the concentration of unreacted GA in the reaction mixture
through UV absorbance at 280 nm assuming that no intramolec-
ular crosslinkings are formed: DC (%) = [(total GA – unreacted
GA)/Mglucosamine] × 100%. where total GA, unreacted GA, and
Mglucosamine are the amount (in moles) of GA in feed, GA unreacted
in reaction mixture, and glucosamine unit in CMCS.
The cross-linked micelles were prepared from NBS–CMCS-1,
NBS–CMCS-2 and NBS–CMCS-3. Their DC was 18.2, 14.5 and 9.6,
respectively.
2.6. Characterization of the micelles
W
LE (%) = (
EE (%) = (
) × 100%
(1)
(2)
W0
The critical micelle concentration (CMC) of amphiphilic
NBS–CMCS in aqueous media was determined by fluorescence
probe technique using Nile red as a hydrphobic probe. A series of
NBS–CMCS-1, NBS–CMCS-2 and NBS–CMCS-3 solutions containing
1 × 10−6 M Nile red were prepared, of which concentration varied
from 5.0 × 10−3 to 0.4 mg/mL. The excitation wavelength (ꢀex) of
Nile red was 550 nm.
The average particle size and size distribution of all the micelle
samples (cross-linked and uncross-linked samples) were deter-
mined by dynamic light scattering (DLS) with a Zetasizer (Malvern
Instruments, Herrenberg, Germany). The measurement was carried
out at a detector angle of 90◦, a wavelength of 512 nm and temper-
ature of 25 ◦C. Morphological evaluation of all the particles was
performed by TEM using a Tecnai G220 (FEI, America).
A − B
) × 100%
A
In Eq. (1), W is the mass of drug in micelles and W0 is the mass of
drug-loaded micelles. In Eq. (2), A is the total amount of the diuron,
and B is the amount of diuron remaining in the supernatant.
The LE and EE of diuron-loaded uncross-linked samples and
cross-linked samples based on NBS–CMCS were determined, and
their values were listed in Table 2.
2.9. Controlled-release of the diuron-loaded micelles
2.7. Photoresponse of the micelles
Lyophilized diuron-loaded uncross-linked or cross-linked
micelle sample (10.0 mg) was dispersed in 4.0 mL of phosphate
buffer solution (PBS) (0.1 M, pH 7.0). The resulting solution was
placed into dialysis bag (cutoff molecular weight: 14,000), then the
dialysis bag was introduced into vial containing 80 mL of PBS (the
same as the solution in the dialysis bag). The system was exposed to
UV light (365 nm, 150 mW cm2) under stirring at 25 ◦C. At appropri-
ate time intervals, 2 mL samples of solution were withdrawn from
the vials and replaced by 2 mL of fresh PBS. The drug release was
assayed by HPLC. The samples without UV light irradiation were
also used as a control.
The photoresponse of the micelles was monitored by DLS
and TEM measurement. Typically, 10.0 mL of aqueous solution of
uncross-linked or cross-linked micelle sample was incubated at
25 ◦C for 5 h and then divided into two equal parts. One part was
irradiated with UV light (365 nm, 150 mW cm−2) for 10 min, the
other part was used as a control and kept in the dark. After that, the
size and morphology of samples were determined by DLS and TEM
measurement.
By solar-simulated radiation, the release of diuron-loaded
micelle samples was also carried out according to the above
method. The used solar simulator is a 300 W Xenon arc lamp
(200–2500 nm spectral output, 50 W radiant output).
2.8. Preparation of diuron-loaded micelles
NBS–CMCS (100 mg) were dissolved in 100 mL deionized water
in a sealed flask. The pH of solution obtained was adjusted to 7.0.

Z. Ye et al. / Carbohydrate Polymers 132 (2015) 520–528
523
Fig. 1. Schematic illustration for the synthesis routes of (A) CMCS, (B) NBS, (C) NBS–CMCS and (D) the assembly process of NBS–CMCS.
The 1H NMR spectrum of NBS–CMCS in DMSO–d6/D2O (1:3, v/v)
is shown in Fig. 2(c). Compared with curve b, the appearance of new
signals in the region of 7.4–8.0 ppm in curve c was the evidence of
benzene ring of NBS. Those at 5.4 ppm and 2.46 ppm correspond to
the benzyl methylene protons (2H, CH2) and the succinic methy-
lene protons (4H, CH2CH2), respectively. The results indicate that
NBS–CMCS have been successfully synthesized.
3. Results and discussion
3.1. Synthesis and characterization of NBS–CMCS
NBS–CMCS was synthesized according to the procedure as
shown in Fig. 1(A–C). During the process, 2-nitrobenzyl succi-
nate (NBS) was firstly prepared by the esterification reaction of
2-nitrobenzyl alcohol with succinic anhydride under the cataly-
sis of DMAP according to the literature (Meng et al., 2013; Yan,
Chen, Tang, & Wang, 2004). Then, CMCS was also synthesized by
the modification of chitosan with ClCH2COOH in alkaline environ-
ment. Finally, NBS was grafted onto the primary amino groups of
CMCS in the presence of EDC and NHS to form NBS–CMCS. Because
of the markedly different water solubility of NBS and CMCS, a mixed
solvent of DMSO/deionized water (1:5, v/v) was used in the process
of grafting reaction. The carboxyl acid group of NBS was first acti-
vated by NHS and then reacted with the primary amino groups in
CMCS to form an amide linkage.
The chemical structures of CS, CMCS and NBS–CMCS were
also determined by FT-IR spectroscopy. Fig. 3 shows their results,
respectively. Curve a shows the basic characteristics of chitosan
at 3455 cm−1 (O H stretch), 2867 cm−1 (C H stretch), 1598 cm−1
(N H bend), 1154 cm−1 (bridge-O-stretch), and 1094 cm−1 (C
O
stretch) (Brugnerotto et al., 2001). Compared with CS (see curve
a), curve b presented new characteristic bands at 1620 cm−1 and
1411 cm−1, which were assigned to COO− antisymmetric and
symmetric stretch. The other characteristic bands of COOH should
be at 3100 cm−1 or so, which were covered by O H hydroxyl
absorption band. Compared with CMCS (see curve b), a new shoul-
der absorption band assigned to the C O stretch of ester group
was observed at 1700 cm−1 in the spectrum of NBS–CMCS (see
curve c). The characteristic bands at 1249 cm−1 and 1055 cm−1
were attributed to the C O stretch of ester group. Besides, the
characteristic bands at 1654 and 1573 cm−1 observably increased
after conjugation, which were attributed to the carbonyl of amide
I band and N H bending of amide II band, respectively. All the
above results confirm the formation of amide linkage between the
carboxyl group of NBS and the amino groups of CMCS.
The 1H NMR spectra of NBS in CDCl3 and CMCS in D2O are
showed in Fig. 2. Their proton signals are assigned, respectively, as
follows (ppm): for NBS (see curve a), 8.12 (d, 1H, ArH), 7.27–7.67 (m,
3H, ArH), 5.56 (s, 2H, ArCH2), 2.75 (s, 4H, CH2CH2), which are iden-
tical with those reported in the previous papers (Meng et al., 2013;
Yan et al., 2004); for CMCS, 2.107 (COCH3), 3.23 (H2), 3.3–3.9 (H3,
H4, H5, H6), 4.460 (H1), 4.524 (-O-CH2-COOD), which are in accor-
dance with the results reported before (Hjerde, Varum, Grasdalen,
Tokura, & Smidsrod, 1997; Chen & Park, 2003).

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Z. Ye et al. / Carbohydrate Polymers 132 (2015) 520–528
Fig. 2. 1H NMR spectra of (a) NBS in CDCl3, (b) CMCS in D2O, (c) NBS–CMCS in DMSO–d6/D2O (1:3, v/v), (d) NBS–CMCS in D2O, and (e) photolytic product in DMSO–d6/D2O
(1:3, v/v) after UV radiation.
3.2. Formation and characterization of micelles
and hydrophilic CMCS backbone. A procedure shown in Fig. 1(D)
provides a description of NBS–CMCS micelle formation. Nile red
was used as a fluorescence probe to monitor the process. If the
polymeric micelles can form in an aqueous solution, Nile red
molecules preferably locate inside the hydrophobic microdomains
of micelles rather than the aqueous phase, resulting in different
Amphiphilic polymer with a proper hydrophilic/hydrophobic
balance can self-assemble into micelle structure in aqueous media.
NBS–CMCS might self-assemble into core–shell micelles because of
its amphiphilic structure comprised of hydrophobic NBS moieties

Z. Ye et al. / Carbohydrate Polymers 132 (2015) 520–528
525
a
b
1620
c
b
1525
1700
1654
1249
d
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber (cm-1
)
Fig. 3. FT-IR spectra of (a) CS, (b) CMCS, (c) NBS–CMCS, and (d) GA cross-linked NBS–CMCS.
photophysical characteristics. As shown in Fig. 5(A), with increase
of NBS–CMCS concentration, the fluorescence emission intensity of
Nile red increased very slowly in low concentration ranges. How-
ever, it increased dramatically at a certain concentration, implying
the formation of micelles and encapsulation of Nile red into the
hydrophobic cores (Kabanov & Alakov, 2002). This concentration
can be defined as a critical micelle concentration (CMC). It is sug-
gested in Fig. 5(A) that the CMC of NBS–CMCS-1 in water was
about 0.057 mg/mL; while those of NBS–CMCS-2 and NBS–CMCS-
3 determined by the same method decreased to 0.026 mg/mL
and 0.019 mg/mL, respectively; this is obviously attributed to the
enhanced hydrophobicity by increase of DS.
The evidence for the formation of the micelles was provided
by TEM experiments. The image (a) in Fig. 4 reveals that there
was obvious contrast between the peripheries and centers in
the spheres, characteristic of the projection images of core–shell
micelles.
The self-assembly behavior of NBS–CMCS was also investigated
by means of 1H NMR. The curve d in Fig. 2 shows 1H NMR spec-
trum of lyophilized NBS–CMCS in D2O. Compared with that of
NBS–CMCS in DMSO–d6/ D2O (see curve c), some signals at 2.46, 5.4,
and 7.4–8.0 ppm ascribed to the protons of appended NBS groups,
especially those signals at 7.4–8.0 ppm assignable to the aromatic
protons, were not observed. It indicated that the hydrophobic
NBS groups in NBS–CMCS was confined within the hydrophobic
domains shielded by hydrophilic CMCS chain segments because
of the formation of shelf-aggregates in D2O, thus leading to pro-
ton signal shielding of NBS. The similar results were also observed
for other amphiphilic polymers that forms micelles in the aqueous
phase (Wang et al., 2011).
tiveness of pesticides. To enhance the micellar structural integrity
and drug encapsulation stability, we selected GA to cross-link the
shell of NBS–CMCS micelles because GA can easily react with amino
groups of NBS–CMCS (Fig. 1D). The FT-IR spectra of NBS–CMCS and
GA cross-linked NBS–CMCS are exhibited in Fig. 3. An absorption
band corresponding to the N H bending at 1525 cm−1 of chains
(see curve d) became very weak in the spectrum of GA cross-linked
micelles. The observation indicates that cross-linking took place
between the amine groups on NBS–CMCS chain and GA. The simi-
lar absorption band has also been observed in the literature (Wang
et al., 2014).
To verify the GA cross-linking further, the concentrations of
micelles were diluted to below CMC. It was found in our experiment
that the uncross-linked micelles were dissociated. However, for
cross-linked micelles, the particles nearly maintained the same size
and PDI value upon high dilution, indicating a successful GA cross-
linking and the ability of cross-linking to stabilize the micelles.
TEM measurements were also taken to evaluate the size and
morphology of GA cross-linked micelles. As shown in Fig. 4(c), the
morphology of cross-linked micelles is similar to that of uncross-
linked micelles (see image a), while the average size decreases from
165 6.3 nm to 140 5.5 nm after cross-linking, showing the for-
mation of more compact structures.
The size and distribution of GA cross-linked micelles were also
measured by DLS (see Table 1). The diameters of cross-linked
samples were smaller than those of uncross-linked. This result is
consistent with that obtained by TEM observation. In addition to the
cross-linking, the hydrophobic interaction also shows the influence
on the nanoparticle size. The diameter of uncross-linked samples
decreased slightly with increasing of DS of NBS, this is obviously
ascribed to that the NBS–CMCS chain with the higher DS of NBS
tends to coil a more shrinking structure. By contrast, a more signif-
icant effect of DS of NBS on the diameter of cross-linked samples
was observed; showing that not only the hydrophobic interaction
but also the cross-linking contributed to this result.
The zeta potential was a useful tool to predict the physi-
cal storage stability of colloidal particles. In our study, the zeta
potential values of all samples were between −25.5 1.24 mV and
−30.5 1.11 mV, which are near the critical value of −30 mV, indi-
cating that all the micelles have good stability. To further confirm
the prediction, GA cross-linked samples were stored for one month
in the dark in the buffer of pH 7.0, their mean diameters and poly-
dispersity index (PDI) values did not change significantly (as shown
3.3. Characterization of shell cross-linked micelles
In pesticide delivery, an important issue need to be consid-
ered is the micelle structural integrity and pesticide encapsulation
stability. When the polymer concentration falls below the crit-
ical micellization concentration (CMC) because of high dilution,
polymeric micelles will dissociate accompanied with undesirable
release of encapsulated pesticides before reaching the targeted
sites (Nakamura, Ryuichi, & Takeda, 1976; Dubey, Jhelum, &
Patanjali, 2011; Jiang, Liu, & Narain, 2009). Moreover, the physi-
cal encapsulation of pesticides within polymeric micelles will lead
to premature pesticide release during storage and reduce the effec-

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Z. Ye et al. / Carbohydrate Polymers 132 (2015) 520–528
Fig. 4. Electron transmission microphotographs of (a) uncross-linked micelles before and (b) after UV irradiation as well as (c) cross-linked micelles before and (d) after UV
irradiation.
in Table 1), whereas the those of the uncross-linked micelles had an
increase to some extent, suggesting that the cross-linking micelles
have higher stability.
material and undergoing expansion as the core domain fills with
water.
In order to further confirm the above speculation, the photo-
response of shell cross-linked micelles was also investigated by
TEM. As expected, the uncross-linked micelles disintegrated upon
UV photolysis due to lack of covalent cross-linking (see image b
in Fig. 4); while the shell cross-linked micelles resulted in a sta-
ble nanocapsule structure after UV irradiation (see image d in
Fig. 4). Their average diameter increased from 140 5.5 nm to
175 6.8 nm. This result is consistent with that obtained by DLS.
The photo-response of NBS–CMCS micelles may be attributed to
photolysis reaction of NBS–CMCS. Fig. 5(B) shows the UV absorp-
tion of the aqueous solution of NBS–CMCS-2 exposed to 365 nm
radiation for different time. The characteristic peak of NBS at
260 nm decreased with increase of the irradiation time and leveled
off after 8 min, indicating that the photolysis reaction took place
under UV stimuli. The 1H NMR from photolytic product in Fig. 2
(see curve e) further supports this result. The signals at 5.4 and
7.4–8.0 ppm ascribed to the protons of 2-nitrobenzyl in NBS were
not observed in curve e, indicating that the phototriggers had been
broken away from NBS–CMCS.
3.4. Photo-responsive property of the shell cross-linked micelles
The photo-responsive properties of the shell cross-linked
micelles were studied through DLS and TEM. As shown in Table 1,
the average diameters of all micelle samples measured by DLS were
between 196 0.11 ∼ 242 0.04 nm at pH 7.0. When the uncross-
linked NBS–CMCS-2 micelle solution was irradiated under UV light
for 10 min, the diameter measured by DLS decreased to about
40.6 nm (see Table 1). This implies that photoremovable side NBS
groups were cleaved from CMCS main chains under UV irradia-
tion, causing a formation of single molecule aggregate (Liu, Chen,
Liu, & Liu, 2008; Chen, Du, Tian, & Sun, 2005). In contrast, the
cross-linked NBS–CMCS-2 micelles allowed for the formation of a
stable structure after photolysis, their average diameter increased
from 196 0.11 nm to 238 0.20 nm. The increase in hydrody-
namic diameter (Dh) after the removal of the hydrophobic core can
be explained by the cross-linked CMCS shell acting like a hydrogel
Table 2
The drug loading efficiency (LE) and encapsulation efficiency (EE) of diuron-loaded uncross-linked micelles and cross-linked micelles based on NBS–CMCS.
Conjugates
Feed ratioa
Uncross-linked micelles
EE (%)
Cross-linked micelles
EE (%)
LE (%)
LE (%)
NBS–CMCS-1
NBS–CMCS-2
NBS–CMCS-3
0.07
0.15
0.26
90.5
95.6
92.3
15.6
37.6
32.3
86.7
91.9
90.5
18.8
40.2
37.5
aMolar ratio of NBS to glucosamine units of CMCS.

Z. Ye et al. / Carbohydrate Polymers 132 (2015) 520–528
527
100
80
60
40
20
0
67
57
47
37
27
17
7
-2.5
-2
-1.5
-1
-0.5
0
0
2
4
6
8
10
LogC (mg/L)
time (h)
1.2
Fig. 6. Release of diuron from the diuron-loaded micelles based on NBS–CMCS-2
in a pH 7.0 buffer under different conditions of light exposure. (ꢁ): uncross-linked
micelles without light irradiation; (ꢀ): shell cross-linked micelles with UV (365 nm,
150 mW cm−2) irradiation (see inset); (᭿): shell cross-linked micelles with solar
simulated irradiation (200–2500 nm spectral output, 50 W radiant output).
no UV
1
1min UV
2min UV
3min UV
6min UV
8min UV
0.8
out UV irradiation, the diuron release from the diuron-loaded shell
cross-linked micelles was not detected, and only 3.35% of the loaded
diuron was released within 8 h for the uncross-linked micelles.
However, with UV irradiation, approximately 45.5% of the loaded
diuron was released from the cross-linked sample within 2 min (see
inset). With the further extension of exposure time to UV light,
the diuron release increased and the cumulative amount was up to
96.9% at 10 min. These results indicate that the shell cross-linked
micelles are able to release the encapsulated diuron in response to
stimuli of UV light.
Sunlight absorption of 2-nitrobenzyl-based chromophores has
already been used to make photo-induced uncaging of bioactive
substances (Sagar, Primavesi, Paul, & Davis, 2014). In order to find
out whether the cross-linked NBS–CMCS micelles can also release
the loaded diuron in response to sunlight, a 300 W Xenon arc lamp
(200–2500 nm spectral output, 50 W radiant output) was used as
solar-simulating radiation for evaluation of the response of the
diuron-loaded cross-linked micelles. A gradual increase in release
rate was observed over 8 h of irradiation (Fig. 6). This result indi-
cates that the broadband can trigger a slow release of diuron from
the cross-linked NBS–CMCS micelles.
0.6
0.4
0.2
0
225
275
325
375
425
475
525
Wavelength(nm)
Fig. 5. (A) Emission intensity of Nile red as a function of the concentrations of
(ꢀ) NBS–CMCS-1, (ꢁ) NBS–CMCS-2 and (—) NBS–CMCS-3. (B) UV–vis spectrum of
NBS–CMCS-2 micelle aqueous solution exposed to 365 nm radiation for different
time.
3.5. Diuron-loading of the shell cross-linked micelles
Core–shell polymeric micelles are particularly suitable for the
encapsulation of hydrophobic active substances because of the
hydrophobic interaction between the substances and the micelle
core (Torchilin, 2004). Diuron is a hydrophobic herbicide, which
is readily encapsulated into micelles in the process of assembly
by mixing with NBS–CMCS solution. Table 2 lists the LE and EE of
diuron-loaded uncross-linked micelles and cross-linked micelles.
The encapsulation efficiency of cross-linked micelles based on
NBS–CMCS-2 reached 91.9%, and the diuron loading efficiency
reached 40.2%, indicating that diuron was efficiently encapsulated
into the micelles. In addition, The LE of uncross-linked micelles was
smaller than that of cross-linked micelles. This may be attributed
to the GA cross-linking, which reduces the leakage of diuron.
4. Conclusions
In our study, a novel amphiphilic conjugate composed of
hydrophilic carboxymethyl chitosan and hydrophobic photola-
bile 2-nitrobenzyl groups was synthesized and its structure was
characterized by FT-IR and 1HNMR. In deionized water the con-
jugate could self-assemble into polymeric micelles through the
hydrophobic interactions between photolabile groups, and further
form cross-linked photoresponsive micelles by dropwise adding
of GA. TEM photograph and 1H NMR investigation showed that the
uncross-linked and cross-linked micelles had a spherical core–shell
structure, which is especially suitable for the encapsulation of
hydrophobic diruon.
3.6. Release studies of the diuron-loaded shell cross-linked
micelles
Research by light scattering showed that the GA cross-linked
micelles in the dark exhibited a high stability and a narrow size
distribution, while an obvious increase in the average diameter
was observed upon exposure to 365 nm UV. An observation by
TEM showed that UV irradiation could trigger the transformation
of the cross-linked micelles into a nanocage struture. The release
from diuron-loaded cross-linked micelles was not detected over
In order to evaluate the potential of the photo-responsive
NBS–CMCS micelles as a controlled release carrier of herbicide, the
release behavior of the diuron encapsulated in NBS–CMCS micelles
in pH 7.0 aqueous solution was studied. As presented in Fig. 6, with-

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Z. Ye et al. / Carbohydrate Polymers 132 (2015) 520–528
8 h without light exposure, while a slow release could be observed
under solar simulated irradiation, suggesting that the cross-linked
micelles may be useful in the delivery of photosynthetic inhibitors
to the sites of photosynthesis, and their further investigations are
in progress.
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Acknowledgments
This research was supported by National Natural Science Foun-
dation of China (No. 51373130) and the Natural Science Foundation
for the Youth (No. 51303145).
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