Glycyrrhetinic acid-modified PEG-PCL copolymeric micelles for the delivery of curcumin

Article abstract of DOI:10.1016/j.reactfunctpolym.2016.12.011

In this study, glycyrrhetinic acid (GA) was conjugated with mono amino poly (ethylene glycol)-b-poly (ε-caprolactone) (H₂N-PEG-PCL) with succinate linker to obtain the GA-modified PEG-PCL (GA-PEG-PCL). By way of thin film hydration method, curcumin was encapsulated into GA-PEG-PCL copolymer to form stable micelles with 93.76% of encapsulation efficiency and 11.93% of drug loading capacity. The micelles enhanced curcumin's water-solubility to 1.87 mg/ml, being 1.70 × 10⁵ times higher than free curcumin. X-ray diffraction and FT-IR analysis confirmed the encapsulation of CUR into copolymeic micelles with an amorphous state. Hemolysis datum of blank micelles showed its biocompatibility. Compared with GA-unconjugated micelles, curcumin-loaded GA-PEG-PCL micelles showed slower in vitro release of drug, but they displayed better in vitro cytotoxicity against HepG2 at about 40 μM because of its selective accumulation in HepG2 cells induced by GA. The results showed that GA-PEG-PCL copolymer could be a promising drug carrier for liver targeted drug delivery.

Full text of DOI:10.1016/j.reactfunctpolym.2016.12.011

Reactive and Functional Polymers 111 (2017) 30–37
Contents lists available at ScienceDirect
Reactive and Functional Polymers
journal homepage: www.elsevier.com/locate/react
Glycyrrhetinic acid-modified PEG-PCL copolymeric micelles for the
delivery of curcumin
a
a
b
a
Runliang Feng a, , Peizong Deng , Zhimei Song , Wei Chu , Wenxia Zhu , Fangfang Teng c,d, Feilong Zhou a
⁎
a
School of Biological Science and Technology, University of Jinan, No. 336 West Road of Nanxinzhuang, Jinan 250022, Shandong Province, PR China
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, No. 15 Datun Road, Chaoyang District, Beijing 100101, PR China
School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, No. l6866 East Road of Jingshi, Jinan 250200, Shandong Province, PR China
Shandong Academy of Medical Sciences, No. 18877 Jingshi Road, Jinan 250062, Shandong Province, PR China
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 April 2016
Received in revised form 11 December 2016
Accepted 14 December 2016
Available online 16 December 2016
In this study, glycyrrhetinic acid (GA) was conjugated with mono amino poly (ethylene glycol)-b-poly (ε-
caprolactone) (H2N-PEG-PCL) with succinate linker to obtain the GA-modified PEG-PCL (GA-PEG-PCL). By way
of thin film hydration method, curcumin was encapsulated into GA-PEG-PCL copolymer to form stable micelles
with 93.76% of encapsulation efficiency and 11.93% of drug loading capacity. The micelles enhanced curcumin's
water-solubility to 1.87 mg/ml, being 1.70 × 105 times higher than free curcumin. X-ray diffraction and FT-IR
analysis confirmed the encapsulation of CUR into copolymeic micelles with an amorphous state. Hemolysis
datum of blank micelles showed its biocompatibility. Compared with GA-unconjugated micelles, curcumin-load-
ed GA-PEG-PCL micelles showed slower in vitro release of drug, but they displayed better in vitro cytotoxicity
against HepG2 at about 40 μM because of its selective accumulation in HepG2 cells induced by GA.
The results showed that GA-PEG-PCL copolymer could be a promising drug carrier for liver targeted drug
delivery.
Keywords:
Curcumin
Poly (ethylene glycol)
ε-Caprolectone
Glycyrrhetinic acid
Micelles
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
controlling the drugs release and improving its bioavailability or activity
[8]. Poly (ethylene glycol)-b-poly (ε-caprolactone) (PEG-PCL) is a well-
Liver cancer, especially hepatocellular carcinoma, is a serious danger
to human health and living. The liver surgery or transplantation is lim-
ited due to cancer multifocality, vascular invasion or shortage of
donor. Chemotherapy is an effective treatment for several cancers be-
cause there are many choices including the use of drugs with different
action mechanism and combination effects of drugs. Curcumin (CUR),
a bioactive component originated from the root of rhizoma curcumae
longae, shows activity against hepatocellular carcinoma in preclinical
experiments via activating reactive oxygen species/TLR-4/caspase sig-
naling pathway [1], inhibiting fatty acid synthase [2], inhibiting hypoxia
inducible factor-1α-induced epithelial-mesenchymal transition [3], and
other mechanisms [4]. It shows no toxicity to normal tissues and organs
[5] with low activity against hepatocellular carcinoma [6]. In addition,
its low water-solubility, non-target, easy metabolism, poor bioavailabil-
ity and pharmacokinetic character also make its application as a drug
limited [7].
known copolymer [9], which can form nanoparticles [10–12], micelles
[13,14], hydrogels [15] and other aggregates [16] to realize the con-
trolled release of CUR, improve its pharmacokinetic property, enhance
its solubility in aqueous medium and its stability. They also show better
[13,17] activity than free drug, or comparable [11] one to it, which is re-
late to its passive target transportation and the release rate without
targeting delivery.
Glycyrrhetinic acid (GA) shows different pharmacological activity
including antiallergy [18], antioxidation [19], liver protection [20,21],
anti-inflammatory [22,23], anticancer [24,25], anti-Addison's disease
[26]. It is noted that the structure of GA has been modified for develop-
ing less toxic chemosensitizing compounds [27–29]. It is recently re-
ported that GA can selectively bond to corresponding receptors of
animal [30] or human [31] liver with high affinity. The chitosan [32–
37] and PEG [38,39] copolymers with GA decoration obviously increase
selective accumulation of drug molecules in liver cells, especially liver
cancers. At the same time, they also achieve drug's controlled release
and promote its activity against cancer.
Polymeric micelles have been widely and deeply studied as drug de-
livery vehicle because of their capability of enhancing hydrophobic drug
water-solubility, passive or active cancer target, stabilizing drug,
In this research, we bounded GA to one side of PEG chain, then PCL to
another side of the chain to afford GA-PEG-PCL copolymer. In order to
enhance CUR's water-solubility and study the effect of GA on the CUR
release behavior from CUR-loaded GA-PEG-PCL (GA-CUR) micelles
⁎
Corresponding author.
E-mail address: runliangfeng@126.com (R. Feng).
http://dx.doi.org/10.1016/j.reactfunctpolym.2016.12.011
1381-5148/© 2016 Elsevier B.V. All rights reserved.

R. Feng et al. / Reactive and Functional Polymers 111 (2017) 30–37
31
and selective transportation of drug-loaded micelles to HepG2 cells, we
synthesized the copolymer and characterized drug-loaded micelles ob-
tained from thin-film hydration procedure. The in vitro cytotoxicity in
various cell lines was then researched.
afford a clear solution and remove undissolved drug and copolymer.
The content of drug in the micelles was determined by UV spectropho-
tometer (New century T6, Beijing Purkinje General Instrument Co., Ltd.,
China) at 425 nm in triplicates [12]. The drug loading capacity (DL) and
encapsulation efficiency (EE) were calculated on the basis of Eqs. (1)
and (2) as follows:
2. Materials and experiments
2.1. Materials
weight of drug in micelles
initial weight of drug
EE ð%Þ ¼
DL ð%Þ ¼
ꢀ 100%
ð1Þ
ð2Þ
CUR was sponsored by Henan Guangye Natural Pigment Co. Ltd.
(Xingyang, China). H2N-PEG2400-PCL was synthesized with monoallyloxy
PEG (Mn = 2400) as raw material according to a process reported for-
merly [14]. Succinic anhydride, N-(3-dimethylaminopropyl)-N′’-ethyl
carbodiimide hydrochloride (EDC) and N-hydroxysuccimide (NHS)
were bought from Sinopharm Chemical Reagent Co. Ltd. (Shanghai,
China). GA was bought from Sigma-Aldrich Co. LLC (St. Louis, MO, USA).
Methylene dichloride was dried with 4 Å molecular sieves. All the other
chemicals and solvents were of analytical grade or higher without further
purification.
weight of drug in micelles
weight of micelles containing drug
ꢀ 100%
The micellar solution or its lyophilized powder was used in the fol-
lowing experiments. The blank GA-modified, -unmodified (blank GA
and blank micelles) and CUR loaded GA-unmodified (CUR micelles) mi-
celles or powder were prepared by the same way without addition of
drug into acetone.
2.2. Synthesis of GA-PEG-PCL
2.4. Characterization of drug-loaded micelles
2.2.1. Preparation of 3-carboxybutyloxy GA
According to a procedure reported previously [34], GA (4.7 g,
10 mmol) and succinic anhydride (2.2 g, 22 mmol) were mixed with an-
hydrous pyridine (80 ml). The reaction mixture was agitated and heated
at 80 °C for 48 h. After removal of solvent, the residue was dissolved in
methylene dichloride (80 ml), washed with hydrochloride (1 mol/l,
20 ml × 3), and extracted with methylene dichloride (30 ml × 3). The
methylene dichloride layers were combined and dried by anhydrous so-
dium sulfate overnight. The solvent was removed on vacuum, and the
residual solid was purified through silica column chromatograph
(hexane:ethyl acetate = 1:1) to afford white powder product with
the yield of 61%. 1H NMR (400M, CDCl3, Bruker BioSpin Corp.): 5.71 (s,
1H, 12-H), 4.55–4.59 (dd, 1H, 3-H), 2.80 (dt, 1H, 1β-H), 2.66 and 2.74
(dt, 4H, suc, –OCOCH2CH2COO–), 2.38 (s, 1H, 9α-H), 1.38 (s, 3H, 27-
CH3), 1.24 (s, 3H, 25-CH3), 1.18 (s, 3H, 29-CH3), 1.14 (s, 3H, 26-CH3),
0.90 (s, 6H, 23- and 24-CH3), and 0.83 (s, 3H, 28-CH3).
2.4.1. Particle size and zeta potentials
The mean particle diameter and zeta potential were measured using
a Zetasizer (3000HS, Malvern Instruments, U.K.) under the same condi-
tion reported previously [14]. The measurement was done in triplicates.
2.4.2. Stability of CUR-loaded micelles
The GA-CUR micelles solution was stored at 4 °C. At 0, 7, 14 and
21 day, the micelles solution was filtered through 0.22 μm millipore fil-
ter. Then drug content in the micellar solution was quantitatively deter-
mined by use of UV spectrophotometer (New century T6, Beijing
Purkinje General Instrument Co., Ltd., China) at 425 nm. And the mean
particle diameter and polydispersity index (PDI) of drug-loaded mi-
celles were also measured in triplicates.
2.4.3. X-ray diffraction and FT-IR spectrum
The X-ray diffraction (XRD) patterns of lyophilized powders from
1.7° to 40° were obtained using X-ray diffractometer (Bruker D8
Focus, Bruker, Germany) with Cu Kα radiation. The FT-IR spectrum of
them from 400 to 4000 cm−1 was recorded by way of KBr pellet method
on a FT-IR spectrometer (Spectrum One, PerkinElmer, USA) at room
temperature.
2.2.2. Preparation of GA-PEG2400-PCL
To a solution of NHS (126.5 mg, 1.1 mmol) and 3-carboxybutyloxy GA
(570 mg, 1 mmol) in methylene dichloride (50 ml), EDC (210.9 mg,
1.1 mmol) was added and stirred at room temperature for 24 h. Then
H2N-PEG2400-PCL2400 (4.8 g, 1 mmol) was mixed with the reaction mix-
ture above followed by continuous reaction at the same temperature for
another 48 h. The most of solvent was removed. The product was obtain-
ed after precipitation from diethyl ether and natural drying. The yield of
target compound was between 78% and 85%. 1H NMR (400M, CDCl3,
Bruker BioSpin Corp.): 4.08 (t, 48H, –OCO-CH2CH2CH2CH2CH2O–), 3.66
(m, 218H, PEG, –OCH2CH2O–), 2.65–2.69 (dt, 4H, suc, –OCOCH2CH2COO–),
2.39 (s, 1H, GA, 9α-H), 2.33 (t, 48H, –OCO-CH2CH2CH2CH2CH2O–), 1.65
(m, 96H, –OCO-CH2CH2CH2CH2CH2O–), 1.44 (m, 48H, –OCO-
CH2CH2CH2CH2CH2O–), 1.38 (s, 3H, GA, 27-CH3), 1.24 (s, 3H, GA, 25-
CH3), 1.18 (s, 3H, GA, 29-CH3), 1.16 (s, 3H, GA, 26-CH3), 0.90 (s, 6H, GA,
23- and 24-CH3), and 0.85 (s, 3H, GA, 28-CH3).
2.4.4. Hemolytic test of GA-PEG-PCL
With rat red blood cell as test sample, hemolytic activities of a series
of concentration of GA-PEG-PCL-2 were investigated according to a pro-
cedure reported previously [40].
2.5. In vitro release of drug-loaded micelles
The in vitro release study of CUR from drug-loaded micelles per-
formed via dialysis technology [14]. In briefly, free CUR, CUR-loaded
GA-unmodified micelles (CUR micelles) and GA-modified (GA-CUR)
micelles containing 1 mg of CUR were added into dialysis bags (Viskas
MD25-3.5, Union Carbide Corporation, Bound Brook, NJ, USA) and put
into containers containing 100 ml of release medium composed of
40 ml of ethanol and 60 ml of saline, respectively. Three parallel samples
were set in each formulation. The containers were oscillated with the
oscillation rate of 300 r/min at 37 °C. At each time point, 5 ml of release
medium was taken. The same volume of fresh release medium was
refilled to make the release medium volume constant. The accumulative
release percentage of CUR was measured according to the reported cal-
culation equation [41] on a UV spectrometer at 425 nm (New century
T6, Beijing Purkinje General Instrument Co., Ltd., China).
Other copolymers containing different length of PCL chain were ob-
tained from the same process. The structure of copolymer was charac-
terized by the 1H NMR with CDCl3 as solvent in NMR spectrometer
(400 MHz, Bruker BioSpin Corp.).
2.3. Preparation of micelles
The copolymer (70 mg) and CUR (10 mg) were co-dissolved in 5 ml
of acetone. The solvent was evaporated on vacuum to form thin mem-
brane, which was hydrated in 5 ml of distilled water at 65 °C, cooled
to room temperature and filtrated through 0.22 μm millipore filter to

32
R. Feng et al. / Reactive and Functional Polymers 111 (2017) 30–37
2.6. Cell uptake experiment
2.8. Statistical analysis
Confocal laser scanning microscopy was used to qualitatively probe
into cellular uptake of CUR, CUR micelles and GA-CUR micelles. HepG2
cells were put into confocal chamber, respectively. The two cell lines
were cultivated overnight before they were co-cultivated with free
CUR dissolved in DMSO (20 μM), CUR micelles (20 μM) and GA-CUR mi-
celles (20 μM). At different time point, cells were photographed by way
of confocal laser scanning microscopy under blue filtered glasses at ex-
citation wavelength of 405 nm.
All experiments were carried out at least in triplicate and results
were expressed as mean S.D. Statistical analysis was performed
using Origin 2015 (Academic) (Originlab Corp., Northampton, MA). Dif-
ference between two groups was analyzed by two sample t-test. Differ-
ence among three or more groups was determined by one-way ANOVA.
Difference with p b 0.05 was considered statistically significant.
In order to investigate quantitatively cellular uptake of free CUR dis-
solved in DMSO (20 μM), CUR micelles (20 μM) and GA-CUR micelles
(20 μM), fluorescent intensity in HepG2 cells was measured after incu-
bation with these formulations, respectively. Briefly, the HepG2 cells
were placed in 96-well plates and cultivated overnight, respectively.
These formulations at a dosage of 20 μM were mixed with the two cell
lines and continuously cultivated for 2 h, respectively. The fluorescent
intensity in each plate was determined by fluorescent microplate at ex-
citation wavelength of 503 nm and emission wavelength of 528 nm, re-
spectively. The experiment was performed in triplicates.
3. Results and discussion
3.1. Synthesis of GA-PEG-PCL
GA-PEG-PCL copolymer was prepared by way of synthetic routine
shown in Fig. 1. GA was firstly transformed into 4-carboxybutyloxy GA
through O-acylation of GA with succinic anhydride. Then selective N-ac-
ylation of H2N-PEG-PCL with NHS and EDC as reaction catalyst and re-
agent afforded GA-PEG-PCL.
Its structure was verified by analysis of 1H NMR listed in Fig. 2. In the
terms of 3-carboxybutyloxy GA, new peaks at 2.66 and 2.74 were the
methylene group's characteristic signals of 3-carboxybutyloxy side
chain [31]. The signal at 3.23–3.28 ppm belonged to proton at H-3 posi-
tion of GA was transferred to 4.55–4.59 ppm when GA was reacted with
succinic anhydride. This confirmed the change of chemical environment
of carbon proton at –OH group after succinic anhydride conjugation. At
the same time, the single peaks of 3-carboxybutyloxy GA at 0.85, 0.90,
1.16, 1.18, 1.24 and 1.38 were assigned to methyl signals being similar
to GA's corresponding groups [27,42]. These data demonstrated that
3-carboxybutyloxy was confessedly introduced into 3-position of GA.
In the 1H NMR of GA-PEG-PCL, new peaks at 0.82–1.27 appeared were
belonged to methyl, methylene and methenyl proton of GA, respective-
ly [27,42]. At the same time, there was a new single peak at 2.19
assigned to amide. Peaks at 1.44, 1.65, 2.33 and 4.08 displayed the meth-
ylene group signals of CL chain in GA-PEG-PCL [43]. The other signals of
2.7. In vitro cytotoxicity test
HepG2 cells were incubated in DMEM containing 10% fetal calf
serum and then transferred into 96-well plates at a density of 5 × 104
per well at 37 °C under 5% CO2 atmosphere. The blank-, drug-loaded mi-
celles and rough drug solution at different concentrations were added
into each well with a ratio of cultural medium to nonmedium equiva-
lent to 9:1 after incubation to be full throughout the wells, respectively.
After incubation for 24 and 48 h, each well was treated with MTT
(5 mg/ml for 4 h, respectively. Then the medium was discarded and to
each well was dimethyl sulfoxide added to dissolve formazan at 25 °C.
Absorbance was determined at 570 nm in Microplate Absorbance Read-
er (Model 680; Bio-Rad Laboratories, Hercules, CA).
Fig. 1. Synthetic routine of GA-PEG-PCL.

R. Feng et al. / Reactive and Functional Polymers 111 (2017) 30–37
33
Fig. 2. 1H NMR spectrum of GA (A), 3-carboxybutyloxy GA (B), GA-PEG-PCL (C) in CDCl3.
GA were so weak that there was no peak observed. These results
showed that the 3-carboxybutyoxyl group of GA was assuredly bonded
to amino group H2N-PE-PCL to form the GA-PEG-PCL. According to the
peak area ratio of signals at 1.25 for methyl of GA and 3.66 for methy-
lene group of PEG, the conjugation efficiency was 96%.
In order to estimate safety of GA-PEG-PCL-2 copolymer, we firstly
processed hemolytic experiment. The hemolysis percentage of copoly-
mer was under 4.11% with its concentration varying from 78 to
2500 μg/ml, showing no obvious hemolytic activity [46]. The hemolysis
percentage was about 7.86% when the concentration of copolymer solu-
tion was 5000 μg/ml. So, the copolymer was biocompatible and could be
used to encapsulate and deliver drug in vivo.
3.2. Characteristics of CUR-loaded micelles
On the basis of EE data above, the CUR-loaded GA-PEG-PCL-2 mi-
celles enhanced CUR water-solubility to 1.87 mg/ml, which was
1.70 × 105 times higher than free CUR [47]. The mean particle diameter
of CUR-loaded GA-PEG-PCL-2 micelles was 40.61 1.54 nm with a PDI
of 0.131 0.023. Transmission electron microscope was applied to ob-
serve and take pictures of CUR-loaded micelles. The picture listed in Fig.
3 displayed that CUR-loaded GA-PEG-PCL micelles were single spherical
particles.
Then the storage stability of drug-loaded micelles was also investi-
gated at 4 °C. The data on EE, DL and particle diameter of drug-loaded
micelles at 0, 7, 14, 21 and 28 days are shown in Table 2. EE and DL hard-
ly changed, indicating that micelles could protect CUR from degradation
or deterioration and keep its high water-solubility. In addition, mean
particle diameter gradually increased with the prolongation of stor-
age-time. This may be related to a dynamic process of micellar disas-
sembly and re-aggregation to generate relatively stable micellar
system [48]. During the whole stability experiment time, the PDI values
of drug-loaded micelles were under 0.3, meaning homogenous size dis-
tribution in the formulation [49,50]. No obvious changes of CUR content
also displayed good and durable encapsulating capacity of drug-loaded
micelles.
PEG and PCL are hydrophilic and hydrophobic polymeric chains in the
PEG-PCL amphiphilic copolymer, respectively. The amphiphilic copoly-
mer self-assembles into micelles with hydrophobic inner core and hydro-
philic outer shell in aqueous circumstance [12]. Hydrophobic PCL will
physically interact with lipophilic drug to encapsulate drug into inner
cores, and longer PCL chains result in its stronger interaction with drug
[44,45]. Meanwhile, hydrophilic PEG outer shell is sufficiently hydrated
to enhance drug's water-solubility. So, weight ratio of PCL to PEG does af-
fect EE and water-solubility of drug-loaded micelles.
The obtained GA-PEG-PCL in this study also contained PCL and PEG
chains. In order to find a micellar formulation having high encapsulating
efficiency and good dispersibility in aqueous circumstance, we selected
PEG2400 as hydrophilic chain and investigated effect of PCL chain num-
ber on the encapsulation of CUR under the condition of fixed PEG
chain length. The results are listed in Table 1. The EE of CUR was about
91.39% when the weight ratio of PCL to PEG was 0.6. Further increase
in weight ratio of PCL to PEG obviously increased EE of CUR to about
93.76%. However, significant precipitant appeared when the weight
ratio of PCL to PEG was raised to 1.8, leading to very low EE (only
about 1.58%), which was related to low water-solubility of GA-PEG-
PCL-3 and CUR. On the basis of the results described above, we selected
GA-PEG-PCL-2 to encapsulate CUR and perform the following
experiments.
Table 1
Encapsulating efficiency and drug loading capacity of GA-CUR micelles.
Copolymer
PEG:PCL (weight ratio)
EE (%)
DL (%)
GA-PEG-PCL-1
GA-PEG-PCL-2
GA-PEG-PCL-3
1:0.6
1:1
1:1.8
91.39
93.76
1.58
0.77
0.80
0.87
11.44
11.93
0.22
0.16
0.06
0.12
Fig. 3. TEM of GA-CUR micelles (100,000, the bar is 50 nm).

34
R. Feng et al. / Reactive and Functional Polymers 111 (2017) 30–37
Table 2
Stability of GA-CUR micelles.
state [40,55], which was helpful for CUR to be easily diffused in
polymeric matrix.
Day
EE (%)
DL (%)
Diameter (nm)
PDI
3.3. In vitro release of CUR-loaded micelles
0
7
14
21
28
94.63
93.19
95.90
94.10
94.10
11.86
11.70
12.00
11.40
11.40
40.61
52.26
71.89
118.81
150.6
1.54
2.01
3.11
4.18
4.821
0.131
0.141
0.116
0.213
0.264
0.023
0.011
0.032
0.041
0.036
The release behavior of drug from its formulation could affect it's
time to effective concentration and pharmacokinetic property. In
order to investigate CUR's in vitro release from GA-PEG-PCL and the
effect of GA on its in vitro release, we selected a dialysis method to per-
form its in vitro release test. In consideration of CUR's low water-solubil-
ity [47] and stability [56], the solution containing ethanol and saline
(2:3, V/V) was selected as the release medium to carry out the release
study [57–59]. The cumulative release plots are provided in Fig. 5. The
free CUR was almost completely released during 6 h. For the two CUR-
loaded micellar formulations, they all slowed down the release of CUR.
The effect of introduction of GA into copolymer on slow release of
curcumin was more obvious than that of GA-unconjugated micelles
during the whole release test. The former resulted in the release of
81.49% of CUR within 12 h, but there were 84.78% of CUR released
from the latter at 10 h.
Folic acid linked with hydrophobic part of MPEG-PCL was found to
partially expose on the surface of the micelles [60]. When it was conju-
gated to hydrophilic part of PEG-PCL, the folate distributed on the sur-
face of micelles [61]. GA is also a lipophilic molecule like folic acid. So,
GA should also locate on the hydrophilic outer shell when GA-PEG-
PCL self-assembled into micelles in aqueous solution. The introduction
of GA made the surface of GA-PEG-PCL micelles more hydrophobic
and difficult to be wet up in comparison with H2N-PEG-PCL micelles
with hydrophilic surface and easy wettability property [62]. This differ-
ence might lead to faster release of CUR from GA-unconjugated H2N-
PEG-PCL micelles (CUR micelles) than corresponding GA-PEG-PCL
micelles.
FT-IR spectrum of raw CUR (a), blank GA micelles (b) and GA-CUR
micelles (c) are shown in Fig. 4. Characteristic combination of a sharp
peak at 3509.7 cm−1 in the free CUR spectrum implied the presence
of aromatic OH group stretching vibrations and the intensive sharp
peaks at 1627.6, 1602.3, 1509.0 and 1428.8 cm−1 corresponding to
mixed C _C, benzene cycle, C _O and olefinic C\\H vibrations [14,
51], respectively, did not interfere with the vibrations in blank micelles
(B) spectra. There were three peaks at 1626.6, 1595.0 and 1515.0 cm−1
similar to peaks at 1627.6, 1602.3 and 1509.0 cm−1 in free CUR spectra
when CUR was loaded into GA-PEG-PCL, which suggested the existence
of CUR in the drug-loaded micelles.
We also performed XRD analysis to determine physical state of CUR
in the drug-loaded micelles. The XRD data are listed in Fig. 4. Blank GA
micelles displayed strong peaks at 19, 21.28 and 23.18° which were dif-
fraction signals of PEG-PCL [52,53] showing a crystalline state of GA-
PEG-PCL. Native CUR showed its characteristic diffraction peaks [54],
but all of them disappeared in the corresponding GA-CUR micelles.
Meanwhile, the diffraction signals of PEG-PCL became very weak.
These results suggested that CUR was in amorphous or solid solution
3.4. Cellular uptake
To investigate whether the enhanced cytotoxicity of our micellar
systems was attributed to the improved intracellular CUR accumulation,
the qualitative analysis of cellular uptake of CUR (a dosage of 20 μM) in
different formulations was examined in HepG2 cells using confocal laser
scanning microscopy (CLSM) at different time points. In Fig. 6, CUR fluo-
rescence signal in HepG2 cells increased with the order of free CUR, GA-
CUR and CUR micelles at different time points, suggesting that CUR was
effectively translocated into the cytoplasm.
To further investigate the uptake efficiency, the intracellular accu-
mulation of CUR was further determined quantitatively by fluorescent
intensity analysis. Fig. 7 shows the fluorescent intensity data generated
Fig. 4. FT-IR (A) and XRD (B) of free CUR (a), blank GA micelles (b) and GA-CUR micelles (c).
Fig. 5. In vitro release of CUR (a), CUR micelles (b) and GA-CUR micelles (c) at 37 °C.

R. Feng et al. / Reactive and Functional Polymers 111 (2017) 30–37
35
Fig. 6. Confocal laser scanning microscopy graph of HepG2 cells treated with free CUR, CUR micelles and GA-CUR micelles at dose of 20 μM at different time point.
from HepG2 cells after incubation for 2 h. In agreement with the confo-
cal study, cells treated with GA-CUR micelles showed mean fluores-
cence intensity (1.87 vs 0.95) in HepG2 cells higher than that treated
with CUR micelles. These data supported the notion that the enhanced
cellular uptake of GA-CUR micelles was specifically mediated by the
GA receptor overexpressed on the HepG2 cells.
that of CUR micelles at 24 h and 48 h at the dose of 40 μM although cel-
lular uptake for free CUR was higher than that for CUR micelles after in-
cubation for 2 h (Figs. 6 and 7). For the three formulations, their
activities against HepG2 were all enhanced at 24 and 48 h when their
dosages were at above 40 μM. Among them, GA-CUR micelles showed
the highest activity, and CUR micelles also displayed better activity
than free CUR. IC50 of free CUR, CUR micelles and GA-CUR micelles
were 59.72, 56.32 and 48.58 μM at 24 h, and IC50 of them were 50.01,
44.97 and 38.62 μM at 48 h, respectively. It has been reported that free
CUR shows low activity at low dose [64]. So, it was inferred that CUR mi-
celles' stronger ability to improve drug bioavailability might play impor-
tant roles in enhancing CUR's activity against HepG2 cells in comparison
with free CUR. More importantly, it is well-known that GA can selective-
ly bond to corresponding GA receptors over-expressed in liver with
high affinity [30,31]. So, on one hand, although the release of CUR
from GA-CUR micelles was slow when compared with that from GA-un-
modified micelles (Fig. 5), introduction of GA into PEG-PCL copolymer
strongly increased cellular uptake of corresponding drug-loaded mi-
celles (Figs. 6 and 7) through GA-mediated targeting delivery to
HepG2 cells. In the first 24 h, the slow CUR release from GA-CUR mi-
celles might surpass GA-mediated uptake, leading to insignificant dif-
ference between the CUR and GA-CUR micelles. However, the GA-CUR
micelles revealed better activity different from crude CUR and CUR mi-
celles after incubation for 48 h. This result should be attributed to more
higher cellular uptake induced by GA compared with CUR micelles,
transcending the slow release of CUR from GA-CUR micelles. On the
other hand, GA-modified micelles might also improve CUR's bioavail-
ability like GA-unmodified micelles in HepG2. These results indicated
that introduction of GA into PEG-PCL copolymer should be beneficial
to improve CUR's activity against HepG 2 cells.
3.5. In vitro cytotoxicity of CUR-loaded micelles
It is reported that CUR shows low therapeutic effect of liver cancer
cells [6]. With the aim of studying whether GA could specifically en-
hance CUR anticancer activity against HepG2 cells, we performed in
vitro cytotoxicity test via MTT method. The results are listed in Fig. 8. Vi-
ability of HepG2 treated with the blank micelles was above 85% [63] at
different concentration during different incubation time, showing that
copolymer had no toxicity against the cell lines.
As for HepG2 cells, there was N78% cells exhibiting viability in cells
exposed to dosage less than or equal to 20 μM in all formulations,
which might be caused by lower concentration than effective concen-
tration [63]. GA-CUR micelles induced the lowest cell viability among
all three formulations (Fig. 8) at a dose of 40 μM. Mean cell viabilities
were about 52% and 48% after incubation with GA-modified micelles
for 24 h and 48 h, respectively. Activity of free CUR was analogous to
4. Conclusion
With the aim of achieving hepatoma cell targeted delivery, GA was
conjugated with monoamino poly (ethylene glycol)-b-poly (ε-
caprolactone) through selective N-acylation of amino group to afford
glycyrrhetinic acid-modified poly (ethylene glycol)-b-poly (ε-
caprolactone) copolymer. The copolymer was biocompatible and encap-
sulated CUR with high EE. The drug-loaded glycyrrhetinic acid-modified
poly (ethylene glycol)-b-poly (ε-caprolactone) micelles could increase
curcumin's water solubility to 1.87 mg/ml and controlled the release of
curcumin. The introduction of glycyrrhetinic acid into monoamino poly
(ethylene glycol)-b-poly (ε-caprolactone) copolymer could obviously
slow down curcumin's release, but it significantly enhanced accumulation
of curcumin in HepG2 cells and resulted in higher in vitro cytotoxicity of
curcumin than glycyrrhetinic acid-unmodified micelles and crude
Fig. 7. Quantitative cellular uptake of free CUR, blank or drug-loaded micelles in HepG2
cells treated at a dosage of 20 μM for 2 h (mean
significant difference (*p b 0.05).
SD, n = 3). Asterisk means a

36
R. Feng et al. / Reactive and Functional Polymers 111 (2017) 30–37
Fig. 8. In vitro cytotoxicity of free CUR, blank and drug-loaded micelles against HepG2 cells after incubation for 24 and 48 h at 37 °C (mean SD, n = 3). Asterisk means a significant
difference (*p b 0.05).
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The authors report no conflicts of interest in this work.
Acknowledgments
This work is supported by a Project of Shandong Province Higher Ed-
ucational Science and Technology Program (J14LM01) and International
Cooperation Training Program of Shandong Province Higher School
Outstanding Young and Middle-aged Backbone Teacher (2013).
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