Self-assembly and β-carotene loading capacity of hydroxyethyl cellulose-graft-linoleic acid nanomicelles

Article abstract of DOI:10.1016/j.carbpol.2016.03.012

A series of linoleic acid conjugated hydroxyethyl cellulose polymers (HEC-g-LA) were synthesized and characterized. And their solubilities in a variety of solvents were compared. The prepared HEC-g-LA polymers showed typical properties of amphiphilic polymers and were able to self-assemble into spherical nanomicelles in aqueous solution. The micelle sizes and critical micelle concentrations (CMC) were found correlated with the molecular structure of polymers, and were varied in the range of 20-50 nm and 1.92-21.76 μg/ml, respectively. Furthermore, the hydrophobic active component β-carotene (β-C) was successfully encapsulated into the HEC-g-LA micelles by sonication-dialysis method. The β-C encapsulation efficiency and loading content were found to be as high as 84.67% (w/w) and 4.23%. The results of in vitro release showed that the encapsulated β-C was continuously released from the micelles in phosphate buffered saline (PBS) medium for about 7 days. The self-assembled HEC-g-LA nanomicelles are potential nanocarriers of hydrophobic active compounds for functional food applications.

Full text of DOI:10.1016/j.carbpol.2016.03.012

Carbohydrate Polymers 145 (2016) 56–63
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Self-assembly and ␤-carotene loading capacity of hydroxyethyl
cellulose-graft-linoleic acid nanomicelles
Yang Yanga, Yanzhu Guoa,b, Runcang Suna,c, Xiaohui Wanga,∗
a State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, PR China
b Liaoning Key Laboratory of Pulp and Paper Engineering, Dalian Polytechnic University, Dalian 116034, PR China
c Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of linoleic acid conjugated hydroxyethyl cellulose polymers (HEC-g-LA) were synthesized and
characterized. And their solubilities in a variety of solvents were compared. The prepared HEC-g-LA
polymers showed typical properties of amphiphilic polymers and were able to self-assemble into spher-
ical nanomicelles in aqueous solution. The micelle sizes and critical micelle concentrations (CMC) were
found correlated with the molecular structure of polymers, and were varied in the range of 20–50 nm
and 1.92–21.76 ␮g/ml, respectively. Furthermore, the hydrophobic active component ␤-carotene (␤-C)
was successfully encapsulated into the HEC-g-LA micelles by sonication-dialysis method. The ␤-C encap-
sulation efficiency and loading content were found to be as high as 84.67% (w/w) and 4.23%. The results
of in vitro release showed that the encapsulated ␤-C was continuously released from the micelles in
phosphate buffered saline (PBS) medium for about 7 days. The self-assembled HEC-g-LA nanomicelles
are potential nanocarriers of hydrophobic active compounds for functional food applications.
© 2016 Elsevier Ltd. All rights reserved.
Received 24 December 2015
Received in revised form 1 March 2016
Accepted 6 March 2016
Available online 9 March 2016
Keywords:
Hydroxyethyl cellulose (HEC)
Linoleic acid (LA)
Nanomicelles
Drug delivery
1. Introduction
tical industry (Donhowe & Kong, 2014; Malaki Nik, Wright, &
Corredig, 2011).
Vitamin A is an indispensable nutrient which plays important
roles in the body including immune function, cell differentiation,
gastrointestinal function and preventing night blindness and so on
(Donhowe & Kong, 2014; Grune et al., 2010). Compared to the other
provitamin A carotenoids, ␤-carotene has the highest vitamin A
activity (Weber & Grune, 2012), which can scavenge reactive oxy-
gen radicals like singlet molecular oxygen, nitrogen dioxide radical,
and peroxyl radical due to the effective antioxidative properties
(Donhowe & Kong, 2014; Lacatusu, Badea, Ovidiu, Bojin, & Meghea,
2012; Weber & Grune, 2012). Moreover, ␤-carotene has been impli-
cated in the prevention or protection against serious human health
disorders such as cancer, heart disease, macular degeneration, and
cataracts(Lacatusu et al., 2012). However, all these beneficial effects
of ␤-carotene are limited due to its poor solubility, high chemi-
cal reactivity and low stability caused by the conjugated polyene
chain(Lacatusu et al., 2012; Xu, Wang, Yuan, Hou, & Gao, 2012). As
a result, ␤-carotene exhibits poor bioavailability and its controlled
delivery remains a challenge for the functional food and nutraceu-
In order to overcome the susceptibility and improve the solubil-
ity and stability of ␤-carotene, some vesicular systems such as noi-
somes (Palozza, Muzzalupo, Trombino, Valdannini, & Picci, 2006),
liposomes(Soppimath, Aminabhavi, Kulkarni & Rudzinski, 2001)
and O/W emulsions(Xu et al., 2012) formed by self-assembling of
different surfactant monomers were investigated(Müller, Petersen,
Hommoss, & Pardeike, 2007). However, the application of the above
systems still have challenges due to their inherent problems such as
low encapsulation efficiency, rapid leakage of water-soluble drug in
the presence of blood components and poor storage stability(Bule,
Singhal, & Kennedy, 2010; Cornacchia and Roos, 2011; Luo, Zhang,
Whent, Yu, & Wang, 2011; Müller et al., 2007; Matalanis, Jones, &
McClements, 2011). Recently, micellar nanoparticles obtained via
self-assembly of amphiphilic polymers have attracted great atten-
tion as nanomedicine carriers for a wide variety of biomedical
applications (Jones & Leroux, 1999; Rösler, Vandermeulen, & Klok,
2012). The polymeric micelles have special core-shell structure, the
hydrophobic inner core of which can solubilize lipophilic drugs
while the hydrophilic shell is responsible for micelles stabiliza-
tion and interactions with plasmatic proteins and cell membranes.
The incorporation of hydrophobic agents into nano-scaled poly-
meric micelles has several advantages. First, polymeric micelles
can readily disperse poorly soluble compounds in aqueous media,
∗
Corresponding author.
E-mail addresses: fewangxh@scut.edu.cn, alicewang1225@163.com (X. Wang).
http://dx.doi.org/10.1016/j.carbpol.2016.03.012
0144-8617/© 2016 Elsevier Ltd. All rights reserved.

Y. Yang et al. / Carbohydrate Polymers 145 (2016) 56–63
57
O
C
N
N
O
+
N
N
HO
CDI
LA
N
O
CO2
+
+
HN
N
N
OH
O
O(CH2CH2O)xH
O
+
O
HEC
O
n
OH
*
OH
O
*
Hx(OH2CH2C)O
OH
OH
O(CH2CH2O)xH
O
O
O
n
OH
*
OH
O
O
*
N
x(OH2CH2C)O
OH
HN
+
O
HEC-g-LA
Fig. 1. The synthesis route of HEC-g-LA copolymers.
and have demonstrated excellent stability both in vitro and in vivo
(Chen, Lee, & Park, 2003; Jones & Leroux, 1999). Second, the nano-
sized structure of polymeric micelles offer them ability to solubilize
poorly soluble agents in large amounts, and achieve site-specific,
controlled delivery as well as substantially increased bioavailabil-
ity of incorporated agents within the micellar interior (Kataoka,
Harada, & Nagasaki, 2012; Sadhukhan, Bakshi, Datta, & Maiti, 2015;
Wiradharma, Zhang, Venkataraman, Hedrick, & Yang, 2009). There-
fore, the polymeric micelles have the potential of overcoming the
limitations of conventional nutraceutical carriers.
homogeneous ring-opening polymerization(ROP) in ionic liquid
and used the obtained nanomicelles as carriers of hydrophobic anti-
tumor drug paclitaxel (PTX). (Guo et al., 2013; Guo, Wang, Shu,
Shen, & Sun, 2012) However, in those direct modifications to cel-
lulose, it is hard to control the hydrophilicity and water solubility
of the amphiphilic cellulose derivatives. It is well known, hydrox-
yethyl cellulose (HEC) is a water soluble cellulose derivative with
enhanced hydrophilicity and reduced crystallinity. Therefore, it is
speculated that using HEC as the starting material to synthesize
amphiphilic cellulose might result in more controllable solubility
and amphiphilic property.
Linoleic acid (LA) is an essential fatty acid for the fatty acid
metabolism in human body (Chen et al., 2003; Jahreis, Kraft,
Tischendorf, Schöne, & von Loeffelholz, 2000). It has been demon-
strated effective in preventing cancer, cardiovascular diseases,
diabetes, and obesity (Belury, 2002; Ferramosca et al., 2006;
Giudetti, Beynen, Lemmens, Gnoni, & Geelen, 2005; House, Cassady,
Eisen, McIntosh, & Odle, 2005; McLeod, LeBlanc, Langille, Mitchell,
& Currie, 2004; Pandey, Renwick, Misquith, Sokoll, & Sparks,
2008). Because of its high hydrophobicity, LA has been success-
fully applied in encapsulation and controlled release of fat-soluble
compounds(Ferramosca et al., 2006).
However, most polymeric micelles are formed by syn-
thetic materials, such as poly(eth ylene glycohol) (PEG) (Topp,
Dijkstra, Talsma,
& Feijen, 1997), poly(N-vinyl-2-pyrrolidone)
(PVP) (Benahmed, Ranger, & Leroux, 2001), poly(vinyl alcohol)
(PVA) (Saito, Okamura, & Ishizu, 1995) and so on. And their appli-
cation in food is limited due to the potential cytotoxicity of the
synthetic polymers (Bagheri, Shateri, Niknejad, & Entezami, 2014).
Therefore, developing biodegradable, nontoxic and biocompati-
ble polymeric micelles derived from natural materials are highly
desired for food industry. (Liu, Jiao, Wang, Zhou, & Zhang, 2008;
Shu et al., 2011; Soppimath et al., 2001). Cellulose is the most
abundant natural and renewable resource on the earth and it
is a promising green material own to its good biocompatibil-
ity, biodegradability, non-toxicity, non-immunogenic (Chen, Tong,
Yang, & Brennan, 2015; Chun-xiang, Huai-yu, Ming-hua, Shi-yu,
& Jia-jun, 2009; Guo, Wang, Shen, Shu, & Sun, 2013). Besides,
cellulose has abundant hydrophilic hydroxyl groups making it
possible to form amphiphilic copolymer by grafting hydropho-
bic segments. Our previous reports have introduce biocompatible
poly(␧-caprolactone) and poly(lactide) into cellulose backbone via
In this paper, a novel amphiphilic cellulose derivative (HEC-g-
LA) was prepared by coupling LA with HEC in order to develop
polymeric nanomicelles suitable for bioactive agents and nutrients
delivery. The HEC-g-LA copolymers show typical amphiphilic prop-
erties and were able to self-assemble into nanoscale capsules. By
using ␤-carotene as a model molecule, the loading and solubilizing
capacities of the HEC-g-LA micelles toward poor soluble nutrients

58
Y. Yang et al. / Carbohydrate Polymers 145 (2016) 56–63
remove CDI and unreacted monomer. The final products were dried
in vacuum oven at 100 ◦C for 24 h.
The yield of the reaction was measured by the weight of HEC.
The formula is showing as follow:
weight of products − weight of origin HEC
WLA =
× 100%
(1)
HEC
weight of origin HEC
2.3. Characterization of HEC-g-LA copolymers
1
2
The molecular structure of HEC-g-LA copolymers was investi-
gated by fourier transform infrared spectroscopy (FTIR) and nuclear
magnetic resonance (NMR) spectroscopy. The FT-IR spectra of pure
HEC and its copolymers were recorded on a Bruker TENSOR 27
spectrophotometer using a KBr disc containing 1% finely ground
3
4
samples at frequencies ranging from 500 cm−1 to 4000 cm−1 1H
.
NMR spectra measurements of HEC and HEC-g-LA samples were
executed on a Bruker Advance 400 MHz spectrometer (AV-600,
Bruker, Germany) operating at 250 MHz, using DMSO-d6 as solvent.
And tetramethylsilane (TMS) was used as an internal standard. The
thermal stability of the HEC-g-LA copolymers was evaluated using
thermogravimetric analysis (TGA Q500, TA, USA), in which samples
were heated in an aluminum crucible at a rate of 10 ◦C/min from
ambient temperature to 600 ◦C, continually flushed with a nitrogen
flow of 25 ml/min.
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber (cm-1)
Fig. 2. FT-IR spectra of unmodified HEC and HEC-g-LA copolymer with different feed
ration of LA. 1–4 corresponds to sample 1–4 in Table 1 respectively.
were evaluated. The in vitro release behaviors of ␤-carotene loaded
HEC-g-LA micelles were also determined.
2.4. Preparation and characterization of HEC-g-LA micelles and
ˇ-carotene loaded HEC-g-LA micelles
2. Experimental
2.4.1. Preparation and characterization of HEC-g-LA micelles
The aqueous solution of HEC-g-LA micelles was prepared by
sonication method using a probe type sonicator (VCX750, Sonics
andmaterials Inc., USA) in an ice bath. In a typical experiment,
10 mg HEC-g-LA copolymer was added into 10 ml ultrapure water.
Afterwards, the solution was ultrasonicated for 30 min.
2.1. Materials
Hydroxyethyl cellulose (HEC, Mw of 250000 g/mol, DS of 1.5, MS
of 2.0), Lionic acid (LA, 98% purity) and N, N’-Carbonyldiimidazole
(CDI, 97.0% purity) were purchased from Aladdin-reagent Inc. HEC
was vacuum dried at 45 ◦C for 48 h before used. ␤-carotene (97.0%
purity) was purchased from Tokyo kasei kogyo Co., Ltd. (Tokyo
Japan). Pyrene (99.0% purity), deuterium oxide (D2O, 100% purity,
99.99 atom% D) and deuterated dimethyl sulfoxide (DMSO-d6,
100% purity, 99.96 atom% D, including 1% (v/v) trimethylsilyl)
were obtained from Sigma-Aldrich Chemical Company, St. Louis,
USA. Dimethyl sulfoxide (DMSO, anhydrous) was purchased from
Sinopharm Chemical Reagent Co., Ltd., China. The dialysis tube
(MWCO 3000 and 7000 Da) was supplied by Shanghai Threebio
Technology Co., Ltd. The copper grid coated with 200 mesh carbon
film was supplied by Beijing Zhongjing Technology Co. Ltd. Phos-
photungstic acid (99.6% purity) was purchased from Tianjin Kermel
Chemical Reagent Co., Ltd. The other reagents, such as acetone and
ethyl alcohol, were of analytical grade and used without further
purification.
The size and size distribution (PDI) of HEC-g-LA micelles were
determined by dynamic light scattering (DLS) on Zetasizer 3000HS
instrument at 90 scattering angle at 25 ◦C. A semiconductor laser
diode (30 mW, 633 nm) was used as the light source. Each test
was repeated at least three times and the average value was cal-
culated. The morphology of HEC-g-LA micelle was obtained by a
field-emission scanning electron microscope (FE-SEM, LEO 1530VP
Germany). TEM images were investigated from a high-resolution
transmission electron microscope (TEM-2100HR, JEOL, Japan) at
an acceleration voltage of 120 kV. The samples for TEM were pro-
cessed as following: the aqueous solutions of HEC-g-LA micelles
that pre-dispersed by ultrasonic treatment were dropped on the
copper wire, and then treated with 2% phosphotungstic acid for
3 min, Finally, the samples were dried in vacuum for 30 min before
testing.
2.2. Synthesis of HEC-g-LA copolymers
2.4.2. Determination of critical micelle concentrations (CMC)
The CMC of HEC-g-LA was determined using a spectrofluo-
rophotometer with pyrene (Sigma, >99%) as the fluorescence probe.
Briefly, the micelle solutions with a series of concentrations in
the range of 1 × 10−4 to 2 mg/ml were initially prepared. The
pyrene/tetrahydrofuran (THF) solution (6.0 × 10−5 M, 50 ␮l) was
added into a 10 ml test tube. And 5 ml micelle aqueous solutions
were added to those test tubes respectively and put in the ultra-
sonic cleaner for 2 h to make pyrene into the core of the micelles.
And then the solvent THF was evaporated under nitrogen. The com-
bined solutions were then kept at room temperature in dark for
24 h in order to reach the solubilization equilibrium of pyrene in the
aqueous system and the final concentration of pyrene in the micelle
solution was controlled to 6.0 × 10−7 M. The fluorescence emission
spectra of pyrene were collected on a Jobin-Yvon Fluorolog Tau-
The LA grafted HEC was synthesized in DMSO without water.
The detailed preparation process for HEC-g-LA copolymers was as
follows: certain amounts of CDI and LA were added into 2 ml DMSO,
and the LA was activated by vigorous magnetic stirring for two
hours at ambient temperature. At the same time, 0.25 g HEC was
dissolved in 5 ml DMSO and stirred for 1 h at 100 ◦C before cooled
down to the room temperature. Then, a transparent solution was
obtained, to which the pre-activated CDI and LA solution was added
dropwise with magnetic stirring. The mixture solution was reacted
for 24 h at room temperature under nitrogen atmosphere. After the
reaction, the resultant copolymers were isolated by precipitating
the mixture with 100 ml ethanol and centrifuged at 4000 rpm for
10 min. Then the residue was washed four times with ethanol to

Y. Yang et al. / Carbohydrate Polymers 145 (2016) 56–63
59
Fig. 3. 1H NMR spectroscopy of HEC-g-LA copolymer (sample 4).
3 system type fluorescence spectrophotometer. The spectra were
recorded from 360 to 550 nm with the excitation wavelength of
339 nm. The excitation and emission slit widths were 5 nm and
2.5 nm, respectively.
solution was transferred into a dialysis tube (MWCO 3000 Da) and
dialyzed against 20 ml phosphate buffer solution (PBS, pH 7.4,
20 mM) which contains 1% sodium salicylate (SDS), with vibrating
in the water-bath shaker at 37 ◦C. After that, 3 ml PBS solution were
taken out timely to measure the concentrations of ␤-C in different
samples by the UV method at 458 nm. The cumulative drug release
was calculated based on the following equation:
2.4.3. Preparation of ˇ-carotene loaded HEC-g-LA micelles
␤-C loaded HEC-g-LA micelles were prepared by sonication-
dialysis method. Firstly, 5 ml HEC-g-LA micelles (1 mg/ml) was
prepared according to the sonication method. Subsequently, a
determined amount of ␤-C dissolved in DMSO solution was slowly
added into HEC-g-LA micelles solution in 10 ml glass tube with
stirring. The mixture was sonicated for 30 min by a probe-type son-
icator (Bandelin SONOPULS, Germany) in an ice bath to make ␤-C
enter into the core of micelles. And then the resultant ␤-C loaded
micelles solution was transferred to a dialysis bag (MWCO = 7000)
and dialyzed against plenty of deionized water for 48 h to remove
DMSO completely. All the solutions obtained were diluted to a cer-
tain volume, followed by filtering using 0.45 ␮m millipore filtration
to remove unloaded ␤-C precipitate and the ␤-C-loaded HEC-g-LA
micelle was finally obtained. With the blank micelles as a control,
the absorbance of ␤-C loaded HEC-g-LA micelles was measured
by UV–vis spectrophotometer at 458 nm. The concentration of ␤-
C was calculated according to a standard curve. The drug loading
content (DL, %) and encapsulation efficiency (EE, %) of ␤-C which
was encapsulated in HEC-g-LA micelles were calculated according
to the following equations:
weightofˇ − Creleasedfrommicelle
Cumulativerelease % =
× 100%(4)
( )
weightofˇ − Cinmicelle
3. Results and discussion
3.1. Synthesis and characterization of HEC-g-LA copolymers
A series of HEC-g-LA copolymers with different contents of
LA were prepared by varying the feeding ratios of LA to HEC.
Fig. 1 illustrates the preparation route of HEC-g-LA copolymer.
The polar organic solvent DMSO was employed to effectively dis-
solve HEC and LA, through which a homogeneous environment for
the reaction was provided. The synthesis of HEC-g-LA copolymer
was completed by an esterification reaction between the hydroxyl
groups of HEC and carboxy groups of LA. However, the esterifica-
tion reaction is reversible, and its reactivity is very low (Hasani &
Westman, 2007). Therefore, activators are essential in this reac-
tion to improve the reactivity of carboxy groups. The commonly
used activators are sodium hydroxide(Ji, Tang, Yan & Sun, 2015),
4-dimethyl aminopyridine(Lu, Li, Tang, Lu & Huang, 2015) (DMAP),
pyridine, sulfuric acid and so on(Sealey, Samaranayake, Todd &
Glasser, 1996), which are not safe for food applications and usu-
ally induce the degradation of cellulose. We used a commercially
available nontoxic activator N,N-Carbonyldiimidazole (CDI) in this
reaction. Comparing with the conventional activators, CDI has
higher reactivity with hydroxyl groups, amino group and carboxy
groups (Armstrong & Li, 2001). In this experiment, CDI was used
to in situ activate carboxy groups of LA to form an intermediate
before the esterification reaction. The intermediate is easy to have
weight of loadedˇ-C
weight of micells
DL(%) =
EE(%) =
× 100%
× 100%
(2)
(3)
weight of loadedˇ-C
weight of totalˇ-C
2.4. Drug release studies in vitro of ˇ-C loaded HEC-g-LA micelles
In vitro ˇ-C release from HEC-g-LA micelle was evaluated using
dialysis method. Typically, 2 ml fresh ␤-C loaded HEC-g-LA micelles

60
Y. Yang et al. / Carbohydrate Polymers 145 (2016) 56–63
Table 1
Synthesis parameters of HEC-g-LA copolymers. HEC:LAa means the molar ratio of HEC to LA, while HEC: CDIb means the molar ratio of HEC and CDI, RT means room
temperature.
Num
HEC:LAa
HEC:CDIb
T ◦
C
WLA
%
CMC (␮g/mL)
Size(nm)
PDI
Solubility
DMSO
Water
1
2
3
4
5
6
7
1:0.25
1:0.5
1:0.75
1:1
1:0.5
1:0.5
1:0.5
1:0.13
1:0.25
1:0.38
1:0.5
1:0.25
1:0.25
1:0.25
R
10.9
21.6
32.5
39.7
25.2
23.8
24.2
21.76
5.73
3.51
1.92
–
102.1
42.8
30.9
25.9
–
0.159
0.387
0.258
0.239
–
+
+
+
+
+
+
+
+
RT
RT
RT
60
80
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
–
–
–
–
–
–
100
“ + ”and “ꢀ” mean soluble and insoluble, respectively.
nucleophilic substitution with the hydroxyl groups of hydroxyethyl
cellulose to form cellulose esters. The carboxylic groups of LA can be
efficiently activated by CDI at room temperature, which avoids pro-
ducing by-products and reduces the degradation of hydroxyethyl
cellulose.Since the degree of polymerization of hydroxyethyl group
in the unit of HEC was uncertain, the weight changes during the
reaction were measured to reflect the reaction efficiency in this
experiment. The weight contents of LA in the HEC-g-LA copolymers
(WLA) were calculated by the gravimetric method and the results
are presented in Table 1. It was found that a higher feeding ratio of
LA could result in a higher weight content of LA in HEC-g-LA copoly-
mer. When the feeding ratio of LA to HEC was increased from 0.25:1
to 1:1, the WLA of HEC-g-LA copolymer was enhanced from 10.9%
to 39.7%. In addition, the aqueous solubility of HEC-g-LA copoly-
mer was also found correlated with the feeding ratio of LA. When
the feeding ratio of LA was below 0.5, the prepared copolymer had
good solubility both in water and in organic solvent. As the feeding
ratio of LA increased, the obtained copolymers were still soluble
in organic solvent, but poorly soluble in water. Furthermore, reac-
tion temperature also has some effects on the water solubility of
HEC-g-LA copolymer. The higher reaction temperature resulted in
reduced solubility of copolymer product, probably due to a higher
degree of substitution was obtained at higher temperature.
Fig. 4. TGA of unmodified HEC and HEC-g-LA copolymer. 1–4 corresponds to sample
1–4 in Table 1 respectively.
were ascribed to the methylene ( CH2 ) of LA. Moreover, the
peak appearing at 1.99 ppm indicated the methylene ( COCH2
)
adjacent to ester group of LA. The 1H NMR spectral investigation
presented in this experiment provides independent evidence for
the successful esterification reaction between LA and HEC, which
was consistent with the result of FT-IR spectra.
The chemical structure of HEC-g-LA copolymers was charac-
terized by FT-IR and 1H NMR techniques. The FT-IR spectra of
unmodified HEC and its grafted derivatives are shown in Fig. 2.
In the spectrum of HEC, the broad peak at 3200–3500 cm−1 was
corresponded to the stretching vibration of OH, where as the peak
at 2700–2900 cm−1 was attributed to the stretching vibration of
To investigate the thermal stability of HEC-g-LA copolymer, TG
test in nitrogen atmosphere were carried out with pure HEC and
graft copolymers, as shown in Fig. 4. It can be seen that when the
weight loss was 50%, thermal degradation temperature of unmod-
ified HEC and samples with the WLA of 10.9, 21.6, 32.5 and 39.7%
were 355, 362, 360, 358 and 367 ◦C respectively. However, when
the weight loss was increased to 80%, thermal degradation tem-
peratures were 377, 389, 396, 408 and 396 ◦C successively. These
results suggested that the thermal stability of HEC-g-LA copolymer
was better than unmodified HEC possibly owing to the high crys-
tallinity of LA. But with the weight content of LA increasing, the
improvement of thermal stability was not obvious. It’s probably
because that the thermal properties of LA esters were not stable as
those of HEC.
CH2 groups. The stretching vibration of C
O C linkage in gluco-
sidic unit appeared at 1109 and 1057 cm−1. Compared with the
spectrum of pure HEC, all of the HEC-g-LA copolymers showed a
new characteristic peak at around 1741 cm−1, corresponding to the
stretching vibration of carbonyl group which suggested the forma-
tion of an ester linkage between HEC and LA. Additionally, the new
peaks at 1453, 1374 cm−1 were observed, which were belong to
the bending and stretching vibration from CH3 and CH2 and in-
plane bending vibration from CH3 of LA. Besides, the peak at around
2925 cm−1 became broader and flatter, indicating that symmetri-
cal and unsymmetrical stretching vibration of CH2 was enhanced.
These results meant that the long alkyl chain was successfully
grafted onto hydroxyethyl cellulose main chain after esterification
reaction. It was also observed that the copolymers exhibit enhanced
absorptions at 1741, 2925 cm−1 with increasing feed ration of LA,
which meant the grafting degree could be controlled by varying
the feed ratio of LA to HEC. These results demonstrated that the LA
side chain was grafted onto HEC successfully, which can be further
confirmed by the 1H NMR results.
3.2. Self-Assembly of HEC-g-LA copolymer
The amphiphilic cellulose micelles characterized by a hydropho-
bic core and a hydrophilic exterior spontaneously form in water
when the polymer concentration is higher than its critical micelle
concentration (CMC). The major driving force behind the self-
association of amphiphilic cellulose is a gain in entropy of the
solvent molecules as the hydrophobic fragments withdraw from
the aqueous surroundings with the formation of micelle core sta-
bilized with hydrophilic segments exposed into water(Guo et al.,
2012). In this experiment, the CMC value of HEC-g-LA copoly-
As shown in Fig. 3. The resonance peaks at 3.05, 3.50, 4.35, 4.55
and 5.31 ppm were due to all kinds of hydrogen in sugar units of
HEC. While the signal at 0.84 ppm was attributed to methyl group
(
CH3 ) in LA. In addition, peaks at 1.02, 1.04, 1.06 and 1.25 ppm

Y. Yang et al. / Carbohydrate Polymers 145 (2016) 56–63
61
1.8
1.6
1.4
1.2
1.0
0.8
1
2
3
4
1E-3
0.01
0.1
1
Concentration (mg/mL)
Fig. 5. The relationship between intensity ratio (I1/I3) of the pyrene emission spectra
and the concentration of HEC-g-LA copolymer. 1–4 corresponds to sample 1–4 in
Table 1 respectively.
mer was investigated by using pyrene fluorescent probe technique.
Pyrene is a molecular fluorescent probe with the intensity ratio of
the first peak (375 nm) and the third peak (386 nm) I1/I3 in its emis-
sion spectrum very sensitive to the polarity of the environment.
When micelles formed, pyrene was preferentially located in the
hydrophobic core of the micelles, and thus the microenvironment
of pyrene changed from hydrophilic to hydrophobic(Guo et al.,
2012). Accordingly, the fluorescence intensity ratio of I1/I3 would
present an obvious change. Fig. 5 exhibits the CMC values of HEC-g-
LA copolymers with different WLA values. The specific CMC values
were showed in Table 1. When the WLA of HEC-g-LA copolymers
was 10.9, 21.6, 32.5 and 39.7%, the CMC values were 21.76, 5.73,
3.51, and 1.92 ␮g/ml respectively. It can be seen that CMC values
decreased with the increased concentration of hydrophobic groups.
It indicated that the increased hydrophobic groups enforced the
hydrophobicity of HEC. As a result, it is easier for the copolymer to
form the micelles, which leads to smaller CMC values.
Besides, the particle size and size distribution of the resulting
self-assembled micelles were evaluated by DLS techniques, the spe-
cific results were exhibited in Table 1. It revealed that the particle
size of the HEC-g-LA micelles with different content of the WLA was
102.1, 42.8, 30.9, and 25.9 nm respectively. It was found that the size
of micelles decreased with increasing the content of LA in HEC-g-
LA copolymers, implying that more hydrophobic substitution can
lead to more compact micelle due to the enhanced hydrophobic
interactions between the LA segments. In addition, Fig. 6(A) and
(B) exhibits the morphology of HEC-g-LA micelles with the WLA of
32.5%. The spherical micelles synthesized by self-assembly were
uniformly distributed in water. And the average size of HEC-g-LA
micelles was range from 20 to 50 nm. The result was corresponded
with DLS analysis, as illustrated in Fig. 6(C).
Fig. 6. (A) and (B):TEM and SEM micrographs of HEC-g-LA micelles.(C) The size
histogram of HEC-g-LA micelles.
Table 2
Characteristic parameters of HEC-g-LA micelles loaded with different ␤-C
concentration.
␤-C/carrier (w/w)
SIZE (nm)
69.0 3.6
PDI
EE (%)
DL (%)
1/20
1/10
1/5
1/2
1/1
0.214
0.19
0.191
0.220
0.145
0.164
0.074
0.009
0.035
0.016
0.025
0.013
84.67
69.90
54.91
44.11
38.50
12.07
4.23
6.99
10.98
22.05
38.5
81.9
127.5
168.5
197.2
212.1
5.8
2.6
9.8
12.6
7.6
2/1
24.14
3.3. Sustained release of ˇ-C encapsulated by HEC-g-LA micelles
In order to improve the solubility and reduce the side-effects
of ␤-C, the aqueous dispersible ␤-C-loaded micelle was pre-
pared by sonication-membrane dialysis method in this study. The
drug-loading parameters and particle sizes of ␤-C loaded HEC-
g-LA micelle with different concentration of ␤-C were shown in
Table 2.The encapsulation efficiency (EE) and drug loading content
(DL) of ␤-C loaded HEC-g-LA micelles were indirectly obtained by
using a UV–vis spectrophotometer at 458 nm. The Table showed
that with the increase of the ␤-C, the drug loading (DL, %) increased
while the encapsulation efficiency (EE, %) decreased. The drug load-
ing content (DL) of ␤-C in HEC-g-LA micelle was increased from
4.23% to 38.5% as the weight ratio of ␤-C was increased from 1/20 to
1/1. However, the DL was decreased dramatically when the weight
ratio of ␤-C change to 2:1. Thus, the concentration of ␤-C is the main
factors to limit the drug loading in initial phase. As the content of ␤-
C increased, ␤-C is saturated in the core, superfluous ␤-C precipitate

62
Y. Yang et al. / Carbohydrate Polymers 145 (2016) 56–63
Fig. 7. The TEM images of ␤-C loaded HEC-g-LA micelles. (␤-C/carrier = 1/2).
Fig. 8. (A) Stability of the blank and the ␤-C encapsulated HEC-g-LA micelles. (B): Cumulative release of ␤-C from HEC-g-LA nanomicelles in PBS.
because of their hydrophobicity, which made the encapsulation
efficiency reduce. Additionally, the sizes of ␤-C-loaded HEC-g-LA
micelles increased from 69.0 nm to 212.1 nm as the weight ratio of
␤-C increased from 1:20 to 2:1, which were mainly caused by the
enlargement in the volume of inner core. It has been established
that the polymeric micelle having size less than 200 nm can effi-
ciently deliver drugs to tumor cells through enhanced permeability
and retention effect(Li et al., 2011). Considering of the size and drug
loading capability, the ␤-C loaded micelles with weight ratio of 1:1
was chosen for the subsequent research of release behaviors.
TEM images of ␤-C loaded HEC-g-LA micelle were shown in
Fig. 7. As shown in Fig. 7A and B, the ␤-C loaded HEC-g-LA micelle
was spherical in shape with a mono-dispersed size distribution.
The sizes of ␤-C loaded HEC-g-LA micelles were about 150 nm,
which was much bigger than the blank micelles. Besides, the sta-
bility of the ␤-C encapsulated HEC-g-LA micelles was investigated,
as showed in Fig. 8(A). Fig. 8(A) showed the average diameters of
the blank and the ␤-C encapsulated HEC-g-LA micelles as time goes
on. It turned out that the change of the diameters of the blank and
the ␤-C encapsulated HEC-g-LA micelles during the ten days with
the room temperature can be ignored. So, the micelles were stable
in room temperature within ten days at least.
or at the interface between the micelle core and the corona were
transferred into the PBS medium. In the next 20 h, the release rate
of ␤-C was obviously slowed down and only about 15.0% of ␤-C
was released in this part. The cumulative release percentage of ␤-C
present a sustainably increased manner between 40 and 150 h, and
about 35.0% of ␤-C was released in this stage. It was also found the
release behavior of ␤-C from drug loaded micelle was stable after
6 days and the cumulative release percentage of ␤-C was 80.0%.
It was noteworthy that about 20.0% of ␤-C was still left in the ␤-
C-loaded micelle after 7 days. This result was probably due to the
increased ␤-C concentration in PBS medium affected its diffusion.
4. Conclusion
In the study, hydroxyethyl cellulose was conjugated with
linoleic acid to make a new amphiphilic HEC-g-LA derivative.
The HEC-g-LA copolymer can form nanomicelles ranging from 20
to 50 nm by self-assembly in water. The HEC-g-LA showed high
dilution stability with very low (1.9 ∼ 21.8 ␮g/mL) CMC. And the
CMC values of HEC-g-LA copolymer decreased with the increasing
content of linoleic acid. Moreover, HEC-g-LA micelles have good
encapsulation efficiency and drug loading ability for hydropho-
bic and antioxidative ␤-carotene, and the ␤-C-loaded HEC-g-LA
micelle showed a sustained release behavior in PBS medium for
about 7 days. In conclusion, a new cellulose based polymeric
surfactant HEC-g-LA was developed as potential nanocarriers of
hydrophobic active compounds for functional food applications.
3.4. In vitro release of ˇ-C from HEC-g-LA micelles
␤-C loaded micelles were prepared by sonication-dialysis
method. The cumulative ␤-C release profiles were shown in
Fig. 8(B). The results showed that ␤-C was gradually released from
the HEC-g-LA micelle for about 200 h. Furthermore, there was an
initial burst release of 20.0% in the first 10 h, which may be owing
to that the ␤-C deposited onto the surface of HEC-g-LA micelles

Y. Yang et al. / Carbohydrate Polymers 145 (2016) 56–63
Jones, M.-C., & Leroux, J.-C. (1999). Polymeric micelles—a new generation of
63
Acknowledgements
colloidal drug carriers. European Journal of Pharmaceutics and Biopharmaceutics,
48(2), 101–111.
This work was financially supported by the Independent Study
Projects of the State Key Laboratory of Pulp and Paper Engineer-
ing (2015C08, 2015ZD03), the Science and Technology Program
of Guangzhou, China(2014J4100039), the New Century Excellent
Talents in University (NCET-13-0215), the Fundamental Research
Funds for the Central Universities, SCUT (201522036) and the
Opening Project of the Key Laboratory of Polymer Processing Engi-
neering, Ministry of Education, China (KFKT-201401).
Kataoka, K., Harada, A., & Nagasaki, Y. (2012). Block copolymer micelles for drug
delivery: design, characterization and biological significance. Advanced Drug
Delivery Reviews, 64, 37–48.
Lacatusu, I., Badea, N., Ovidiu, O., Bojin, D., & Meghea, A. (2012). Highly antioxidant
carotene-lipid nanocarriers: synthesis and antibacterial activity. Journal of
Nanoparticle Research, 14(6), 1–16.
Li, Y., Heo, H. J., Gao, G. H., Kang, S. W., Huynh, C. T., Kim, M. S., et al. (2011).
Synthesis and characterization of an amphiphilic graft polymer and its
potential as a pH-sensitive drug carrier. Polymer, 52(15), 3304–3310.
Liu, Z., Jiao, Y., Wang, Y., Zhou, C., & Zhang, Z. (2008). Polysaccharides-based
nanoparticles as drug delivery systems. Advanced Drug Delivery Reviews,
60(15), 1650–1662.
Lu, Q.-l., Li, X.-y., Tang, L.-r., Lu, B.-l., & Huang, B. (2015). One-pot tandem reactions
for the preparation of esterified cellulose nanocrystals with
4-dimethylaminopyridine as a catalyst. RSC Advances, 5(69), 56198–56204.
Luo, Y., Zhang, B., Whent, M., Yu, L., & Wang, Q. (2011). Preparation and
characterization of zein/chitosan complex for encapsulation of ␣-tocopherol,
and its in vitro controlled release study. Colloids and Surfaces B: Biointerfaces,
85(2), 145–152.
References
Armstrong, A., & Li, W. (2001). N,Nꢀ-Carbonyldiimidazole. In Encyclopedia of
reagents for organic synthesis. John Wiley & Sons, Ltd.
Bagheri, M., Shateri, S., Niknejad, H., & Entezami, A. (2014). Thermosensitive
biotinylated hydroxypropyl cellulose-based polymer micelles as a nano-carrier
for cancer-targeted drug delivery. Journal of Polymer Research, 21(10), 1–15.
Belury, M. A. (2002). Inhibition of carcinogenesis by conjugated linoleic acid:
potential mechanisms of action. The Journal of Nutrition, 132(10), 2995–2998.
Benahmed, A., Ranger, M., & Leroux, J.-C. (2001). Novel polymeric micelles based
on the amphiphilic diblock copolymer
poly(N-vinyl-2-pyrrolidone)-block-poly(d,l-lactide). Pharmaceutical Research,
18(3), 323–328.
Bule, M. V., Singhal, R. S., & Kennedy, J. F. (2010). Microencapsulation of
ubiquinone-10 in carbohydrate matrices for improved stability. Carbohydrate
Polymers, 82(4), 1290–1296.
Chen, N., Tong, Z., Yang, W., & Brennan, A. B. (2015). Biocomposites with tunable
properties from poly(lactic acid)-based copolymers and carboxymethyl
cellulose via ionic assembly. Carbohydrate Polymers, 128, 122–129.
Chen, X.-G., Lee, C. M., & Park, H.-J. (2003). O/W emulsification for the
self-Aggregation and nanoparticle formation of linoleic acid modified chitosan
in the aqueous system. Journal of Agricultural and Food Chemistry, 51(10),
3135–3139.
Müller, R. H., Petersen, R. D., Hommoss, A., & Pardeike, J. (2007). Nanostructured
lipid carriers (NLC) in cosmetic dermal products. Advanced Drug Delivery
Reviews, 59(6), 522–530.
Malaki Nik, A., Wright, A., & Corredig, M. (2011). Micellization of beta-carotene
from soy-protein stabilized oil-in-water emulsions under In vitro conditions of
lipolysis. Journal of the American Oil Chemists’ Society, 88(9), 1397–1407.
Matalanis, A., Jones, O. G., & McClements, D. J. (2011). Structured
biopolymer-based delivery systems for encapsulation, protection, and release
of lipophilic compounds. Food Hydrocolloids, 25(8), 1865–1880.
McLeod, R. S., LeBlanc, A. M., Langille, M. A., Mitchell, P. L., & Currie, D. L. (2004).
Conjugated linoleic acids, atherosclerosis, and hepatic very-low-density
lipoprotein metabolism. The American Journal of Clinical Nutrition, 79(6),
1169S–1174S.
Palozza, P., Muzzalupo, R., Trombino, S., Valdannini, A., & Picci, N. (2006).
Solubilization and stabilization of ␤-carotene in niosomes: delivery to cultured
cells. Chemistry and Physics of Lipids, 139(1), 32–42.
Chun-xiang, L., Huai-yu, Z., Ming-hua, L., Shi-yu, F., & Jia-jun, Z. (2009). Preparation
of cellulose graft poly(methyl methacrylate) copolymers by atom transfer
radical polymerization in an ionic liquid. Carbohydrate Polymers, 78(3),
432–438.
Cornacchia, L., & Roos, Y. H. (2011). State of dispersed lipid carrier and interface
composition as determinants of beta-carotene stability in oil-in-water
emulsions. Journal of Food Science, 76(8), C1211–C1218.
Pandey, N. R., Renwick, J., Misquith, A., Sokoll, K., & Sparks, D. L. (2008). Linoleic
acid-enriched phospholipids act through peroxisome proliferator-activated
receptors ␣ to stimulate hepatic apolipoprotein A-I secretion. Biochemistry,
47(6), 1579–1587.
Rösler, A., Vandermeulen, G. W. M., & Klok, H.-A. (2012). Advanced drug delivery
devices via self-assembly of amphiphilic block copolymers. Advanced Drug
Delivery Reviews, 64(Suppl. (0)), 270–279.
Donhowe, E., & Kong, F. (2014). Beta-carotene: digestion, microencapsulation, and
in vitro bioavailability. Food and Bioprocess Technology, 7(2), 338–354.
Ferramosca, A., Savy, V., Conte, L., Colombo, S., Einerhand, A. W. C., & Zara, V.
(2006). Conjugated linoleic acid and hepatic lipogenesis in mouse: role of the
mitochondrial citrate carrier. Journal of Lipid Research, 47(9), 1994–2003.
Giudetti, A. M., Beynen, A. C., Lemmens, A. G., Gnoni, G. V., & Geelen, M. J. H. (2005).
Hepatic lipid and carbohydrate metabolism in rats fed a commercial mixture
of conjugated linoleic acids (Clarinol G-80TM) 1. European Journal of Nutrition,
44(1), 33–39.
Grune, T., Lietz, G., Palou, A., Ross, A. C., Stahl, W., Tang, G., et al. (2010). ␤-Carotene
is an important vitamin a source for humans. The Journal of Nutrition, 140(12),
2268S–2285S.
Guo, Y., Wang, X., Shen, Z., Shu, X., & Sun, R. (2013). Preparation of
cellulose-graft-poly(␧-caprolactone) nanomicelles by homogeneous ROP in
ionic liquid. Carbohydrate Polymers, 92(1), 77–83.
Guo, Y., Wang, X., Shu, X., Shen, Z., & Sun, R.-C. (2012). Self-assembly and paclitaxel
loading capacity of cellulose-graft-poly(lactide) nanomicelles. Journal of
Agricultural and Food Chemistry, 60(15), 3900–3908.
Hasani, M., & Westman, G. (2007). New coupling reagents for homogeneous
esterification of cellulose. Cellulose, 14(4), 347–356.
House, R. L., Cassady, J. P., Eisen, E. J., McIntosh, M. K., & Odle, J. (2005). Conjugated
linoleic acid evokes de-lipidation through the regulation of genes controlling
lipid metabolism in adipose and liver tissue. Obesity Reviews, 6(3), 247–258.
Jahreis, G., Kraft, J., Tischendorf, F., Schöne, F., & von Loeffelholz, C. (2000).
Conjugated linoleic acids: physiological effects in animal and man with special
regard to body composition. European Journal of Lipid Science and Technology,
102(11), 695–703.
Sadhukhan, S., Bakshi, P., Datta, R., & Maiti, S. (2015). Poly(ethylene oxide)-g-gellan
polysaccharide nanocarriers for controlled gastrointestinal delivery of
simvastatin. Journal of Applied Polymer Science, 132(32), n/a–n/a.
Saito, R., Okamura, S., & Ishizu, K. (1995). Synthesis of poly(vinyl alcohol)
core-polystyrene shell type microspheres. Polymer, 36(23), 4515–4520.
Sealey, J. E., Samaranayake, G., Todd, J. G., & Glasser, W. G. (1996). Novel cellulose
derivatives. IV. Preparation and thermal analysis of waxy esters of cellulose.
Journal of Polymer Science Part B: Polymer Physics, 34(9), 1613–1620.
Shu, S., Sun, L., Zhang, X., Wu, Z., Wang, Z., & Li, C. (2011). Polysaccharides-based
polyelectrolyte nanoparticles as protein drugs delivery system. Journal of
Nanoparticle Research, 13(9), 3657–3670.
Soppimath, K. S., Aminabhavi, T. M., Kulkarni, A. R., & Rudzinski, W. E. (2001).
Biodegradable polymeric nanoparticles as drug delivery devices. Journal of
Controlled Release, 70(1–2), 1–20.
Topp, M. D. C., Dijkstra, P. J., Talsma, H., & Feijen, J. (1997). Thermosensitive
micelle-Forming block copolymers of poly(ethylene glycol) and
poly(N-isopropylacrylamide). Macromolecules, 30(26), 8518–8520.
Weber, D., & Grune, T. (2012). The contribution of ␤-carotene to vitamin A supply
of humans. Molecular Nutrition & Food Research, 56(2), 251–258.
Wiradharma, N., Zhang, Y., Venkataraman, S., Hedrick, J. L., & Yang, Y. Y. (2009).
Self-assembled polymer nanostructures for delivery of anticancer
therapeutics. Nano Today, 4(4), 302–317.
Xu, D., Wang, X., Yuan, F., Hou, Z., & Gao, Y. (2012). Stability of ␤-carotene in
oil-in-water emulsions prepared by mixed layer and bilayer of whey protein
isolate and beet pectin. Journal of Dispersion Science and Technology, 34(6),
785–792.
Ji, B., Tang, P., Yan, K., & Sun, G. (2015). Catalytic actions of alkaline salts in
reactions between 1,2,3,4-butanetetracarboxylic acid and cellulose: II
esterification. Carbohydrate Polymers, 132, 228–236.