Linolenic acid-modified PEG-PCL micelles for curcumin delivery

Article abstract of DOI:10.1016/j.ijpharm.2014.05.059

In this study, a novel linolenic acid-modified poly(ethylene glycol)-b-poly(E-caprolactone) copolymer was prepared through radical addition, ring-opening polymerization, and N-acylation reactions. Its structure was characterized by ¹H NMR and GPC. Micelles were developed by thin-film hydration and used as a delivery system for curcumin with high drug loading capacity of 12.80% and entrapment efficiency of 98.53%. The water solubility of curcumin was increased to 2.05 mg/mL, which was approximately 1.87 × 10⁵ times higher than that of free curcumin. The micelles were spherical shape with an average diameter of 20.8 ± 0.8 nm. X-ray diffraction and FT-IR studies suggested that curcumin existed in the polymeric matrices under π-π conjugation and hydrogen bond interaction with the copolymer. In vitro drug release studies indicated that the curcumin release from linolenic acid-modified copolymer micelles met controlled release, and its release rate was less than that from the linolenic acid-unmodified copolymer micelles. Cytotoxicities against Hela and A-549cells demonstrated that the additional π-π conjugation could affect curcumin's anticancer activity through reducing its release from micelles. Hemolysis test and intravenous irritation test results revealed that the linolenic acid-modified copolymer was safe for intravenous injection. The plasma AUC_0-∞ and MRT_0-∞ of curcumin-loaded linolenic acid-conjugated poly(ethylene glycol)-b-poly(E-caprolactone) copolymer micelles were 2.75- and 3.49-fold higher than that of control solution, respectively. The CL_z was also decreased by 2.75-fold. So, this linolenic acid-modified copolymer might be a carrier candidate for curcumin delivery.

Full text of DOI:10.1016/j.ijpharm.2014.05.059

International Journal of Pharmaceutics 471 (2014) 312–321
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Linolenic acid-modified PEG-PCL micelles for curcumin delivery
Zhimei Song a, Wenxia Zhu b,c, Na Liu a, Fengying Yang a, Runliang Feng a,
*
a
Department of Pharmaceutical Engineering, School of Biological Science and Technology, University of Jinan, 336 Nanxinzhuang Xilu, Jinan, Shandong
Province 250022, PR China
b
School of Medicine and Life Sciences, University of Jinan, 336 Nanxinzhuang Xilu, Jinan, Shandong Province 250022, PR China
Shandong Academy of Medical Sciences, 18877 Jingshi Road, Jinan, Shandong Province 250062, PR China
c
A R T I C L E I N F O
A B S T R A C T
Article history:
In this study, a novel linolenic acid-modified poly(ethylene glycol)-b-poly(e-caprolactone) copolymer
Received 10 January 2014
Received in revised form 1 May 2014
Accepted 29 May 2014
was prepared through radical addition, ring-opening polymerization, and N-acylation reactions. Its
structure was characterized by 1H NMR and GPC. Micelles were developed by thin-film hydration and
used as a delivery system for curcumin with high drug loading capacity of 12.80% and entrapment
efficiency of 98.53%. The water solubility of curcumin was increased to 2.05 mg/mL, which was
approximately 1.87 ꢀ105 times higher than that of free curcumin. The micelles were spherical shape with
an average diameter of 20.8 ꢁ 0.8 nm. X-ray diffraction and FT-IR studies suggested that curcumin existed
Available online 2 June 2014
Keywords:
Linolenic acid
Curcumin
PEG-PCL
in the polymeric matrices under
p–p conjugation and hydrogen bond interaction with the copolymer. In
vitro drug release studies indicated that the curcumin release from linolenic acid-modified copolymer
micelles met controlled release, and its release rate was less than that from the linolenic acid-unmodified
Micelles
copolymer micelles. Cytotoxicities against Hela and A-549cells demonstrated that the additional
p–p
conjugation could affect curcumin’s anticancer activity through reducing its release from micelles.
Hemolysis test and intravenous irritation test results revealed that the linolenic acid-modified copolymer
was safe for intravenous injection. The plasma AUC0–1 and MRT0– of curcumin-loaded linolenic acid-
1
conjugated poly(ethylene glycol)-b-poly(e-caprolactone) copolymer micelles were 2.75- and 3.49-fold
higher than that of control solution, respectively. The CLz was also decreased by 2.75-fold. So, this
linolenic acid-modified copolymer might be a carrier candidate for curcumin delivery.
ã 2014 Published by Elsevier B.V.
1. Introduction
administration, drug targeting to the specific issues, solubility of
hydrophobic drugs (Grottkau et al., 2013). Poly(ethylene glycol)
Curcumin (CUR) is a low-molecular-weight, natural polyphe-
nolic compound isolated from the rhizome of turmeric (Curcuma
(PEG), a hydrophilic polymer, can avoid to phagocytosis of reticulo-
endothelialsystem(RES)and preventhydrophobiccompoundsfrom
the absorption of blood proteins to prolong their cycling time in vivo
(Wang et al., 2011; Wei et al., 2012; Zhang et al., 2011). Hydrophobic
longa). It has
a low intrinsic toxicity with anti-oxidative,
-inflammatory, -microbial, and -tumor properties (Maheshwari
et al., 2006; Ono et al., 2004). Preclinical studies of CUR have
shown its ability to inhibit a variety of cancer cell lines (Aggarwal
et al., 2003). The ability of CUR to induce apoptosis in cancer cells
without cytotoxic effects on healthy cells makes it a potential
compound against cancer (Aggarwal et al., 2003; Hatcher et al.,
2008). However, its clinical potential has been greatly limited due
to extremely low solubility in aqueous solution (2.99 ꢀ10ꢂ8 M) as
well as its poor bioavailability (Letchford et al., 2008).
poly(
D
,
L
-lactide) (PLA) (Tu et al., 2007), poly(
e-caprolactone) (PCL)
(Jia et al., 2008), poly(
d
-valerolactone) (Nair et al., 2011) (PVL), and
poly(glycolide)(PGA)(Dingetal.,2012)havebeencoupledwithPEG
to form amphiphilic copolymer, respectively. These copolymers are
biodegradable and biocompatible, which have distinct hydrophilic
and hydrophobic segments forming an inner core and outer shell
due to their large solubility difference in aqueous medium. They
have been used to encapsulate lipid drugs under hydrophobic
interactionstoenhancedrug’ssolubilityoractivity(Gaoetal., 2009;
Hu and Zhang, 2010), change drug’s crystalline state (Shin et al.,
2010; Zhang et al., 2010b), and improve controlled release property
(Derakhshandeh et al., 2010; Hu and Zhang, 2010). For example,
methoxy PEG-PCL diblock copolymer can successfully encapsulate
CURwithahigherdrugloadingcapacity, improvingCUR’ssolubility,
Micelles are extensively researched as drug delivery systems,
which have several advantages such as small size particles, ease of
* Corresponding author. Tel.: +86 531 89736825; fax: +86 531 89736818.
E-mail address: runliangfeng@126.com (R. Feng).
http://dx.doi.org/10.1016/j.ijpharm.2014.05.059
0378-5173/ã 2014 Published by Elsevier B.V.

Z. Song et al. / International Journal of Pharmaceutics 471 (2014) 312–321
313
pharmacokinetics property or anticancer activity (Chen et al., 2011;
2.3. Synthesis of H2N-PEG-PCL diblock copolymer
Gong et al., 2013; Kanazawa et al., 2011; MaLing et al., 2011; Shao
et al., 2011; Yin et al., 2013).
The H2N-PEG-PCL diblock copolymer was synthesized through
a previously published method of MPEG-PVL copolymer (Lee et al.,
2005) and PCL-PPG-PCL preparation (Oh et al., 2009). To remove
the last traces of water, H2N-PEG (12.4 g, 5 mmol) was azeotroped
with toluene (50 mL) at 120 ꢃC for 4 h in a 100 mL three-neck flask.
The toluene was removed by evaporation. To the residual
dicloromethane (200 mL), 1 mol/L hydrochloride ethyl ether
It is noted that modification of PEG with long-chain fatty acid
also forms a typical core-shell type structure for encapsulating
clonazepam into inner core and showing controlled release (Lee
et al., 2003). Furthermore, linolenic acid (LNA) has several
conjugated double bonds which can interact with aromatic cycle
or other conjugated bond via p–p orbital overlapping (Heard et al.,
2005). Consequently, compared with PEG-PCL without LNA (NH2-
PEG-PCL), it is believed that LNA-modified PEG-PCL (LNA-PEG-PCL)
(10 mL) and different weight of e-CL were added in turns. The
reaction mixture was stirred at 25 ꢃC for 24 h. The resultant
mixture was evaporated under vacuum and precipitated in 100 mL
of diethyl ether. The H2N-PEG-PCL diblock copolymer was obtained
after filtration and drying under atmosphere. 1H NMR (CDCl3):
can afford additional
CUR, a polyphenolic compound containing a large conjugation
system. This additional orbital overlapping interaction should
p–p orbital overlapping interaction with
p–p
strengthen the encapsulation of CUR into the inner core in aqueous
medium.
d
1.33–1.43(48H, m, OOCꢂꢂCH2CH2CH2CH2CH2O), 1.56–1.70(96H,
m, 2.29–2.34(48H, m,
OOCꢂꢂCH2CH2CH2CH2CH2O),
The main objective of this study is to investigate the
OOCꢂꢂCH2CH2CH2CH2CH2O), 2.5–2.95(6H, m, NH2CH2CH2ꢂꢂS-
CH2CH2CH2CH2ꢂꢂO), 3.65(218H, m, OCH2CH2O), 4.04–4.08(48H,
t, OOCꢂꢂCH2CH2CH2CH2CH2O).
feasibility of LNA-PEG-PCL micelles as
a drug carrier for
anticancer drugs. This copolymer was synthesized and charac-
terized by 1H NMR and GPC. Its biocompatibility was evaluated
by use of hemolysis test. Curcumin’s physical state and in vitro
release kinetics from the LNA-PEG-PCL micelles were studied.
We also performed in vitro cytotoxicity tests to investigate
effects of LNA modification of PEG-PCL on cytotoxicity against
Hela and A-549cancer cell lines.
2.4. Synthesis of LNA-PEG-PCL diblock copolymers
The ethyl linolenate (3.06 g, 10 mmol) dissolved in methanol
(50 mL) was mixed with 5 mol/L sodium hydroxide (4 mL,
20 mmol) and stirred for 24 h at room temperature. The solvent
was evaporated under vacuum. The residue was dissolved in
methylene dichloride, washed with water to neutral, dried with
anhydrous sodium sulfate and evaporated in vacuum to obtain
LNA.
2. Materials and methods
2.1. Materials
LNA (556 mg, 2 mmol) and N,N-dicyclohexyl carbamide
(494 mg, 2.4 mmol) were dissolved in methylene dichloride
(30 mL) and agitated for 0.5 h at room temperature. To the
mixture, N,N-dimethylamine pyridine (12.2 mg, 0.1 mmol) and
different molecular weight of H2N-PEG-PCL (1.8 mmol) were added
and continuously stirred for 24 h at the same temperature. The
reaction mixture was filtered to remove formed precipitate,
evaporated under vacuum to dry and precipitated with ethyl
CUR was purchased from Fluka Chemical Company Inc. (Buchs,
Switzerland). Allyloxy poly(ethylene glycol) (APEG) (Mn = 2400)
was procured from Sungder Chemical (Nantong) Co., Ltd. (Nantong,
Jiangsu Province, China) and dried twice by azeo-distillation of
toluene. e-Caprolactone (e-CL) was provided by Huayuan polymer
Co. Ltd. (Qingdao, Shandong Province, China). Cysteamine hydro-
chloride purchased from Xinhaihong Chemical Industry Co., Ltd.
(Hangzhou, Zhejiang Province, China) was dried by use of silica gel
in desiccators for 48 h. 2,20-Azobisisobutyronitrile (AIBN) was
bought from Damao Chemical Reagent Factory (Tianjin, China).
Methylene dichloride and N,N-dimethyl formamide (DMF) were
dried by 4 A molecular sieves. Ethyl linolenate was bought from
Linuo Biochemistry Co., Ltd. (Anyang, Henan Province, China). All
the other chemicals and solvents were of analytical grade or higher,
obtained commercially.
ether to gain LNA-PEG-PCL. 1H NMR (CDCl3):
d0.95–1.00(3H, t,
CH3CH2 of LNA), 1.31–1.43(58H, m, OOCꢂꢂCH2CH2CH2CH2CH2O),
1.60–1.70(106H, m, OOCꢂꢂCH2CH2CH2CH2CH2O of PCL and 3-CH2
to 7-CH2 of LNA), 2.07(4H, t, 8-CH2 and 17-CH2 of LNA), 2.28–2.33
(50H, m, OOCꢂꢂCH2CH2CH2CH2CH2O of PCL and 2-CH2 of LNA),
2.82(4H, m, 11-CH2 of LNA and NHCH2CH2-SCH2), 3.64(218H, m,
OCH2CH2O of PEG), 4.04–4.08(48H, t, OOCꢂꢂCH2CH2CH2CH2CH2O),
4.22(2H, t, terminal CH2 of PEG).
2.5. Preparation of micelles
2.2. Synthesis of H2N-PEG
Micelles were prepared by thin-film hydration (Wang et al.,
2012). CUR and copolymer (1:7 (w/w)) were co-dissolved in
acetone in a round-bottomed flask to form a clear solution. A
yellowish thin layer of uniform film on the wall of flask formed
under vacuum rotary evaporation. The resulting thin film was
hydrated in 5 mL water with moderate rotating at 65 ꢃC, filtered
The H2N-PEG was synthesized based on a methodology
reported about H2N-PEG-N3 (Hiki and Kataoka, 2007).
A
500 mL three-neck flask equipped with a magnetic stirring bar
was charged with APEG2400 (12 g, 5 mmol), cysteamine hydro-
chloride (11.35 g, 100 mmol), AIBN (0.82 g, 5 mmol), and 250 mL of
dried DMF. The mixture was stirred at 65–70 ꢃC for 24 h under
nitrogen flow. The solvent was removed by rotary evaporation
under vacuum. The residual solid was treated with 1 mol/L
potassium carbonate solution (100 mL), evaporated to dry,
dissolved in methylene dichloride (150 mL), and filtered in turns.
The filtrate was concentrated to 1/10 of the initial volume. After
precipitated from an excess volume of diethyl ether, the H2N-PEG
through a 0.22 mm filter membrane to remove non-incorporated
drugs and copolymer aggregates and used for further analysis or
lyophilization. Bank micelles were prepared in a similar approach.
The accurate volume of drug-loaded micelles solution was
added into ethanol in a 10 mL of volumetric flask to disrupt the
micelles’ core-shell structure and dissolve CUR releasing from the
micelles. Through stepwise dilution, a solution of CUR with UV
absorbance at a range of 0.2–0.8 was provided. The CUR content in
the drug-loaded micelles was determined using the UV–vis
spectrophotometer at 425 nm. The drug loading content (DL)
and drug encapsulation efficiency (EE) were calculated based on
the following formula (Song et al., 2011):
polymer was obtained (10.9 g, yield 88%). 1H NMR (CDCl3):
d1.82–
1.91(2H, m, SꢂꢂCH2CH2CH2ꢂꢂO), 2.64(2H, t, SꢂꢂCH2CH2CH2ꢂꢂO),
2.75(2H, t, NH2CH2CH2ꢂꢂS-CH2CH2CH2CH2ꢂꢂO), 3.03 (2H, t,
NH2CH2CH2ꢂꢂS-CH2CH2CH2CH2ꢂꢂO), 3.39–3.89 (218H, m, OCH2-
CH2O).

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Z. Song et al. / International Journal of Pharmaceutics 471 (2014) 312–321
Weight of drug in nanoparticles
Weight of the initial drug
containing the released hemoglobin (Hb), was collected. The
EE ¼
ꢀ 100%
absorbance of Hb in the supernatant at 500–650 nm was measured
by UV–vis spectrophotometer (T6 New Century, Purkinje General,
Peking, China).
The degree of hemolysis was determined on the basis of
absorbance at 576 nm and calculated from the following formula-
tion (Dutta and Dey, 2011):
Weight of drug in nanoparticles
Weight of nanoparticles containing drug
DL ¼
ꢀ 100%
2.6. Charaterization of micelles’ physico-chemical properties
AbssampleꢂAbsnegative control
2.6.1. Morphology of CUR-loaded LNA-PEG-PCL (CUR-LNA) micelles
Transmission electron microscope (TEM, JEM-1200EX, JEOL,
Tokyo, Japan) was used for morphological observation. One drop of
micelles solution was placed on a carbon film-coated copper grid of
400 meshes. After negative staining with 2% phosphotungstic acid
for 20 s, the excess solution was absorbed by filter paper and dried
in air before analysis.
Hemolysisð%Þ ¼
ꢀ 100%
Abspositive controlꢂAbsnegative control
where Abssample, Absnegative, and Abspositive were absorbance of
copolymer sample, physiological saline (0% hemolysis) and
distilled water (100% hemolysis). All assays were performed in
triplicate.
2.6.5. Intravenous irritation test
2.6.2. Particle size and zeta potential of blank LNA-PEG-PCL (blank
LNA) and CUR-LNA micelles
One New Zealand rabbit with weight of 2.0–2.5 kg was injected
with a daily dose of 15 mg/kg of CUR-LNA micelles solution and
equivalent volume of physiological saline into the right and left
ear-border vein at an injection rate of 1 mL/min for 3 days,
respectively (Jing et al., 2014). After injection, pathological reaction
at the injection site was recorded. The rabbits were sacrificed by
bleeding 24 h after the last administration. At the localizations of
1.3 cm from the injection site to proximal part, the ears were cut
and fixed in 10% formalin for histological observation.
Particle size and its distribution (expressed as polydispersity
index, PDI) of blank LNA and CUR-LNA micelles were determined
by dynamic light scattering (DLS, Zetasizer 3000HS, Malvern
Instruments Ltd., UK). The micelles were suspended in deionized
water before measurement under a fixed scattering angle of 90ꢃ at
25 ꢃC in the condition of He–Ne laser (633 nm). The surface charge
of the drug-free and -loaded micelles suspension in deionized
water was estimated by Laser Doppler Anemometry (Zetasizer
3000HS, Malvern Instruments Ltd., UK). Results were expressed as
mean ꢁ standard deviation (SD). All the measurements were
analyzed in triplicates.
2.7. In vitro release of CUR-LNA micelles
In vitro drug release profile of CUR from CUR-LNA micelles was
done by dynamic dialysis method previously reported with a
solution of ethanol-normal saline (40:60) as release medium(Sun
et al., 2012). CUR-LNA micelles and CUR-loaded NH2-PEG-PCL
micelles (CUR micelles) control solution containing 1 mg of CUR
were put into dialysis bags (Viskas MD25-3.5, Union Carbide
Corporation, Bound Brook, NJ, USA) with a 3500 Da molecular cut-
off, respectively. The bags were placed into 100 mL release
mediums in a beaker and vibrated with 100 rpm/min of oscillation
at 37 ꢃC. At definite time intervals, 5 mL of the release mediums
was withdrawn. The same volume of fresh release mediums was
supplemented to maintain a constant volume. The release amount
of drug was quantified spectrophotometerically at a wavelength of
425 nm. All experiments were performed in triplicates. The release
rate was calculated from the following equation, and the results
were expressed as mean ꢁ SD:
2.6.3. X-ray diffraction (XRD) and FT-IR studies of CUR, blank LNA and
CUR-LNA micelles
XRD analysis was used to confirm if CUR exists in the
crystallographic form in the nanoparticulate CUR formulation or
not. The XRD patterns of CUR, blank LNA and CUR-LNA micelles
were obtained from X-ray diffractometer measurements (Bruker
D8 Focus, Bruker, Germany). The measurements were performed at
a voltage of 40 kV and 25 mA using Cu K
a radiation. The scanned
angle was set from 1.7ꢃ to 40ꢃ, and the scan rate was 2ꢃ/min.
FT-IR spectra were also done to research the possible physical
and chemical interactions between the CUR and the polymer
matrix. FT-IR spectrum of the CUR, lyophilized blank LNA, and CUR-
LNA micelles was obtained by KBr pellets method on a FT-IR
spectrometer (Spectrum One, PerkinElmer, USA), respectively. The
wavenumber range was set from 4000 to 400 cmꢂ1
.
Qn ¼ CnV0 þ nꢂ1CiVi
X
i¼0
2.6.4. Hemolysis test of copolymers
Hemolysis assay was performed using a method formerly
elaborated to evaluate the copolymer’s biocompatibility (Meng
et al., 2011). Briefly, heparinized rat erythrocytes were separated
from 5 mL of rat blood by centrifugation at 3000 rpm for 30 min in a
centrifuge (TG16-WS, Xiangyi instrument Co. Ltd., Xiangtan,
Hunan Province, China) and washed with physiological saline to
achromatic color for supernatant solution. 1 mL of the purified
erythrocytes was mixed with 39 mL of normal saline to form a 2.5%
erythrocytes suspension (v/v). 1.6 mL of the resulting suspension
was mixed with 4 mL of copolymers’ solution in normal saline
prior to shaking incubation for 30 min at 37 ꢃC in an oscillator
(SHZ-88, Jintan Medical Instrument Factory, Jintan, Jiangsu
Province, China). The copolymer concentration range was set
Qn
W
Rð%Þ ¼
ꢀ 100%
where Qn is the accumulative released amount of drug, Cn is the
drug concentration in the release medium of each time interval, V0
is the total volume of the release medium, Vi is the volume of the
withdrawn medium, Ci is the drug concentration in the release
medium at time i, R is the abbreviation of accumulative release
percentage, and W is the total drug weight in each dialysis bags.
2.8. In vitro cytotoxicity of micelles
The HeLa and A-549cells were cultured in Dulbecco’s modified
Eagle’s medium supplemented with 10% fetal bovine serum (FBS)
in 24-well plate. Then, all cells were incubated for 24 h in incubator
(37 ꢃC, 5% CO2). The culture medium was removed, and fresh
couture medium containing different dosages of CUR, blank
micelles, CUR micelles, blank LNA micelles, or CUR-LNA micelles
from 156 to 2500 mg/mL. Physiological saline and distilled water
were used as negative and positive control, respectively. After
incubation, the samples were centrifuged for 20 min at 8000 rpm/
min for the purpose of isolating unbroken erythrocytes and
disrupted membranes from the solution. The supernatant,

Z. Song et al. / International Journal of Pharmaceutics 471 (2014) 312–321
315
was added each well, respectively. Cells were co-incubated at 37 ꢃC
for 48 h. The drug dosages were set as 0, 5, 10, 20 and 40 mol/L,
3. Result and discussion
m
respectively. The blank copolymer concentration was the same as
the corresponding curcumin-loaded micelles. The culture medium
without treatment was used as control group. For cytotoxicity
assay after incubation for 48 h, the culture medium containing
3.1. Synthesis and characterization of LNA-PEG-PCL
In a former article, the H2N-PEG-PCL diblock copolymer is
synthesized by radical addition reaction of cysteamine hydrochlo-
ride and allyl-PEG-PCL under AIBN as catalyst (Feng et al., 2013).
However, there may be ammonolysis side-reaction of PCL chain in
this reaction. In this article, H2N-PEG was firstly prepared from
radical addition reaction of APEG with cysteamine hydrochloride,
then the pure H2N-PEG-PCL diblock copolymer was synthesized
micelles was displaced by 90
mL of fresh culture medium, and
10 L of MTT solution (5 mg/mL) was added to each well, and the
m
plates were incubated for 4 h. The medium was removed, and the
MTT formazan crystals were then dissolved with 1 mL of DMSO at
ambient temperature. The optical density was measured at 490 nm
with
a
spectrophotometer (Multiskan GO 1.00.40, Thermo
through ring-opening copolymerization reaction of e-CL with H2N-
Scientific). The viable rate was determined by comparing the
absorption at 490 nm with control wells containing only cell
culture medium (Rezaei et al., 2012). The experiments were
performed in triplicates.
PEG because amino group showed different reaction activity from
hydroxyl group in H2N-PEG under hydrochloride diethyl ether as
catalyst (Fig. 1). The LNA-PEG-PCL was prepared from N-acylation
reaction of H2N-PEG-PCL diblock copolymer with LNA in condition
of DCC as condensation reagent and DMAP as catalyst (Fig. 1).
The 1H HMR spectrum of H2N-PEG was listed in Fig. 2A; the
signals were clearly observed at 1.82–1.91 (d, OCH2CH2CH2S), 2.62
(c, OCH2CH2CH2S), 2.75 (b, SCH2CH2NH2), and 3.03 ppm (a,
CH2SCH2CH2NH2) corresponding to the resulting structure via
the radical addition of 2-aminoethanethiol to allyl group of APEG
shown as Fig. 1. The corresponding signals were similar to that of
H2N-PEG-N3 reported by Hiki (Hiki and Kataoka, 2007).
2.9. Pharmacokinetics of CUR-LNA micelles
Animal experiments were conducted in full compliance with
national regulatory principles. Male Sprague–Dawley rats
(250 ꢁ 20 g) were supplied by the center for new drug
evaluation Shandong University (Shandong, China). The rats
were housed under standard conditions of temperature,
humidity and light, and took standard rodent diet and water
freely ahead of each experiment. The rats fasted overnight prior
to drug dosing.
Free CUR was completely dissolved in a solution being made up
of N,N-dimethyl acetamide (15%, v/v), PEG-400 (45%, v/v) and 5%
glucose solution (40%, v/v) to afford CUR control (Song et al., 2011).
Two groups of rats (six/group) were given CUR control or CUR-
LNA micelles at 15 mg/kg via the tail veins injection, respectively.
After drug administration, blood samples were withdrawn into
heparinised centrifugal tubes and centrifuged at 3000 rpm/min for
10 min for plasma separation at the definite time. The gathered
plasma was stored at ꢂ20 ꢃC prior to analysis. The treatment of
plasma samples was performed according to a method described
previously (Song et al., 2011). A Shimadzu LC-10A HPLC system
equipped with a UV detector was used to determine quantitatively
the concentration of CUR in plasma samples at 425 nm. The mobile
phase contained a 52% acetonitrile and 48% citric acid buffered
solution (pH 3.0, v/v). The flow rate was set at 1 mL/min. The
The LNA-PEG-PCL was analyzed by 1H HMR (Fig. 2C). The peak
at 0.95–1.00 (j) originated from methyl group of linolenyl, and the
peaks at 1.60–1.70 (o–s), 2.07 (k + n), 2.28–2.33 (t) and 2.82 (m)
were assigned to methylene groups of linolenyl at 3- to 7-, 8-, 17-,
2- and 11-positions, respectively (Fig. 2C), which were simliar to
that of LNA reported early (Crombie and Morgan, 1991). The
characteristic GPC curve of LNA-PEG2400-PCL2400 is shown as Fig. 3.
The monopeak indicated that the LNA-PEG-PCL copolymeer was
prepared without any other side reactions.
3.2. Characterization of micelles
The LNA-PEG-PCL copolymer contained hydrophilic PEG and
hydrophobic PCL or LNA. It is thought that the copolymer can also
self-assembly form a core-shell structure in aqueous medium. The
hydrophilic core’s interaction with hydrophilic drug results in
increasing drug’s water-solubility, but its percentages in the
copolymer have important effect on improving drug’s water
solubility when the molecular weight of PEG is constant (Lee et al.,
2005; Sun et al., 2013). So, in order to gain an ideal drug
formulation, we researched the effect of PCL chain length on
curcumin’s encapsulation efficiency (EE) when molecular weight
injection volume for all samples was 5 mL.
The plasma concentration vs. time profile for each rat was
analysed using DAS 2.0 software to evaluate the pharmacokinetic
parameters via non-compartmental model analysis.
Fig. 1. Synthesis scheme of LNA-PEG-PCL.

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Fig. 2. 1H HMR spectrum of H2N-PEG (A), H2N-PEG-PCL (B) and LNA-PEG-PCL (C).

Z. Song et al. / International Journal of Pharmaceutics 471 (2014) 312–321
317
Fig. 4. TEM of CUR-LNA micelles (19000ꢀ, the scale bar is 60 nm).
micelles showed strong peaks at 2931 and 2864 cmꢂ1 attributing
to the stretching vibrations of trans-alkene CꢂꢂH bonds LNA chain.
Peaks at 1735 and 1702 cmꢂ1 corresponded to the stretching
vibrations of ester carbonyl and amide carbonyl groups, and a peak
at 1657 cmꢂ1 was due to CQC stretching of LNA chain. The
CH2ꢂꢂOꢂꢂCH2 stretching vibration and LNA’s trans-alkene CꢂꢂH
deformation were found at 1108 and 1298 cmꢂ1, respectively. In
micellar formulation, the wave numbers corresponding to the
characteristic peaks of LNA-PEG-PCL were shifted due to the
complexation of CUR with the LNA-PEG-PCL NPs. Comparing LNA-
PEG-PCL with drug loaded LNA-PEG-PCL micelles, peak shifts were
observed from 3448 to 3414 cmꢂ1, 1735 to 1734 cmꢂ1, 1657 to
1628 cmꢂ1, and 1298 to 1284 cmꢂ1. The blue shift of 1735–
1734 cmꢂ1 should be attributed to hydrogen bond between CUR
and LNA-PEG-PCL copolymer (Xie et al., 2011; Yen et al., 2010).
The FT-IR spectrum of CUR exhibited an absorption band at
3509 cmꢂ1 attributed to the phenolic OꢂꢂH stretching vibration
(Yallapu et al., 2010). Additionally, sharp absorption bands at
1627 cmꢂ1 (stretching vibration of CQC bond), 1602 cmꢂ1
(stretching vibrations of benzene ring), 1509 cmꢂ1 (CQO and
CꢂꢂC vibrations), 1429 cmꢂ1 (olefinic CꢂꢂH bending vibration), and
1280 cmꢂ1 (aromatic CꢂꢂO stretching vibration) were similar to
characteristic peaks of curcumin reported previously (Yallapu
et al., 2010). These sharp absorption bands were also shifted to
1628, 1589, 1515, 1454, and 1284 cmꢂ1 when CUR was loaded into
the LNA-PEG-PCL micelles, respectively. The blue shift of stretching
Fig. 3. GPC of LNA-PEG2400-PCL2400
.
of PEG was constant. As shown in Table 1, the EE was 96.60 ꢁ 2.16%
for LNA-PEG2400-PCL1200, which was similar to LNA-PEG2400
-
PCL2400, but the former copolymer was unstable and precipitated
when stored in 4 ꢃC refrigerator. By contrast, the EE obviously
decreased for LNA-PEG2400-PCL4800. This might be relative to the
copolymer’s hydrophobic properties and interaction with drug.
The hydrophobic interaction would be in favor of increasing CUR’s
solubility in water, but the length of hydrophobic PCL must be
suitable. The CUR’s solubility in water would be decreased when
the length of PCL exceed a limitation. The LNA-PEG2400-PCL2400
was selected to be studied in depth on the basis of higher EE.
In this study, UV–vis spectrophotometer was selected for
quantitative analysis of CUR (without results shown). The total
volume of CUR-NP solution was 4.8 mL. The DL and EE of CUR-LNA
micelles prepared from LNA-PEG-PCL were 12.80% and 98.53%,
respectively. So the water solubility of CUR was increased to
2.05 mg/mL, which was 1.87 ꢀ105 times higher than that of CUR in
water (Letchford et al., 2008).
The mean particle diameters of blank and CUR-loaded LNA-
PEG2400-PCL2400 micelles measured from the dynamic light
scattering were 19.5 ꢁ 2.1 nm and 20.8 ꢁ 0.8 nm, respectively.
The drug was loaded into micelles, resulting in an increase on
the mean particle diameter. Zeta potentials of blank and CUR-
loaded LNA-PEG2400-PCL2400 micelles were ꢂ2.63 ꢁ 0.17 mV and
ꢂ2.12 ꢁ 0.09 mV, respectively. This result indicated that the
micelles were nearly neutral. Surface morphology of the CUR-
loaded LNA-PEG2400-PCL2400 micelles was examined by TEM. Fig. 4
shows the TEM image of the CUR-LNA micelles. The particles
seemed to have a spherical shape and a smooth surface. The TEM
image further confirmed the particle size detected from the
dynamic light scattering.
FT-IR spectroscopy was performed to ascertain the formation of
CUR-LNA micelles. Fig. 5 shows the FT-IR spectrum of CUR, blank
LNA and CUR-LNA micelles. The FT-IR spectrum of blank LNA
Table
1
Characterization of CUR-LNA micelles.
Copolymer
Molecular weight from GPC EE
(%)
DL
(%)
LNA-PEG2400
PCL1200
LNA-PEG2400
PCL2400
LNA-PEG2400
PCL4800
-
10,649
19,823
24,926
96.60 ꢁ 2.16 11.96 ꢁ 0.39
98.53 ꢁ 1.73 12.80 ꢁ 0.16
53.04 ꢁ 0.19 6.63 ꢁ 0.24
-
-
Fig. 5. FT-IR spectrum of CUR, blank LNA micelles and CUR-LNA micelles.

318
Z. Song et al. / International Journal of Pharmaceutics 471 (2014) 312–321
concentrations from 156 to 2500 mg/mL. The hemolytic assay
indicated that LNA-PEG2400-PCL2400 showed no obvious hemolytic
activity (hemolysis percentage <5%) (Cerda-Cristerna et al., 2011).
After the 3-day injection treatment of CUR-LNA micelles
solution or physiological saline, no erythema and congestion of
blood vessel were found in CUR-LNA micelles under the observa-
tion by naked eye. The microscopical observation demonstrated
that vascular line and endotheliocyte structures were clear and
intact, and no tissue edema and thrombus were found (Fig. 8).
Furthermore, inflammatory cell infiltrate was not observed in the
vessel wall and surrounding tissues. For physiological saline
control, there were also no histophatological changes (Fig. 8). All
the results demonstrated that CUR-LNA micelles solution at a dose
of 15 mg/kg show no stimulus response in the ear vein of rabbit.
3.3. In vitro release of CUR-LNA micelles
The in vitro drug release curves of the CUR-LNA and CUR-loaded
NH2-PEG2400-PCL2400 micelles (CUR micelles) are shown in Fig. 9.
Due to the low solubility of CUR in water (Letchford et al., 2008),
40% (v/v) of ethanol saline solution was used as the dialysis
medium to provide a sink condition. The drug release percentage
Fig. 6. XRD of CUR, blank LNA micelles and CUR-LNA micelles.
vibration of CUR’s benzene ring (1602–1589 cmꢂ1) and LNA’s CQC
(1657–1628 cmꢂ1) was due to extension of conjugated bond caused
by their
p–p orbital overlapping interaction. These interactions
may have effects on the in vitro or in vivo release and uptake of drug
(Heard et al., 2005), and then on its activities.
XRD studies were performed to investigate the physical state of
CUR in the micellar particles, because this would impact the in vitro
and in vivo drug release from the micellar formulations. Fig. 6
shows the XRD diagrams of pure CUR, CUR-LNA micelles and blank
LNA micelles. The pure CUR peaks at angles 2u appeared at a series
of diffraction at 8.84ꢃ, 12.14ꢃ, 14.46ꢃ, 17.26ꢃ, 18.12ꢃ, 21.14ꢃ, 23.36ꢃ,
24.50ꢃ, 25.54ꢃ, 27.34ꢃ, 28.14ꢃ, and 28.96ꢃ, which indicated that pure
CUR was in a highly crystalline form (Donsi et al., 2010; Yen et al.,
2010). However, these characteristic peaks did not exist in CUR-
LNA micelles, showing that the state of CUR was amorphous,
disordered crystalline phase or in solid solution (Anitha et al.,
2011a,b,b; Zhang et al., 2010b). It is beneficial to excellent diffusion
of drug molecules through the polymeric matrix, leading to a
sustained release of the encapsulated drug.
Hemolysis assay will give additional information about this
copolymer’s biocompatibility in the case of in vivo application
(Zobel et al.,1999). The profiles of hemolysis assay of LNA-PEG2400
-
PCL2400 copolymer were shown in Fig. 7. The LNA-PEG2400-PCL2400
exhibited hemolytic activity from 1.10% to 5.81% at the copolymer
Fig. 8. Microscopical observation of vascular irritability study with different
formulations of CUR-LNA micelles (A) and physiological saline (B) after
administration in rabbits.
Fig. 7. Hemolysis percentage of LNA-PEG2400-PCL2400 at different concentrations.

Z. Song et al. / International Journal of Pharmaceutics 471 (2014) 312–321
319
have any obvious effect on proliferation of Hela and A-549. In other
words, polymer materials had no any activities and effect on the
cytotoxicity determination of CUR loaded in micelles. In the MTT
experiment, CUR-LNA micelles were compared with CUR micelles
to determine if LNA affected CUR’s anticancer activity or not. Two
kinds of CUR-loaded micelles obviously decreased the viability of
Hela cells (below 48%) when the concentrations of CUR in NP were
20
function against A-549 only when CUR’s concentration reached to
40 mol/L (Fig. 10) at which the viability rate was below 48%.
mmol/L or above (Fig. 10), while they showed obvious killing
m
Certainly, CUR control showed higher activity than both of the
CUR-loaded micelles in all dosages. This should be related to
slower sustained release of CUR from micelles (Tao et al., 2013;
Wang et al., 2012). The slower release resulted in lower
concentration in cells, which showed inferior activities in
comparison with CUR control. It was interesting that CUR micelles
had higher activities against cancer cells than that of CUR-LNA
micelles. This phenomenon should also be caused by more slower
release of CUR from CUR-LNA micelles in which there were two
stronger hydrogen bond (Xie et al., 2011; Yen et al., 2010) and
p–p
Fig. 9. In vitro CUR-release profile from CUR and CUR-LNA micelles in a solution of
ethanol-normal saline (40:60) as release medium at 37 ꢃC (data are presented as
mean ꢁ SD (n = 3)).
orbital overlapping (Heard et al., 2005) afforded by PEG-PCL and
LNA, respectively. These interactions were confirmed by FT-IR
spectrum shown in Fig. 5, and in vitro release results also
supported that there was a causal relationship between release
and anticancer activity.
from the CUR and CUR LNA micelles was found to be 78.78% and
60.84% of the encapsulated drug during the first 12 h, respectively,
while the release percentage of CUR reached 84.69% and 81.3%
within following 12 h, respectively. The different release rates
indicated that linolenic acid chain might interact with CUR,
slowing CUR’s release from CUR-LNA micelles. The initial faster
drug release of micelles should be attributed to fraction of CUR
loacated (or closed to) on the surface of micelles (Zhang et al.,
2010a). It made CUR release faster from the LNA micelles.
3.5. Pharmacokinetics of CUR-LNA micelles
Sprague–Dawley rats were used to study pharmacokinetics of
CUR-LNA micelles by intravenous route, which was also compared
with CUR control solution. Fig. 11 shows the blood concentration–
time curves, and Table 2 lists typical pharmacokinetic parameters
of the two preparations. As shown in Fig. 11, CUR could not be
detected after 1.5 h for CUR control while it could continue to exist
during 6 h for CUR-LNA micelles.
3.4. In vitro cytotoxicity of micelles
The plasma concentration – time curves for CUR control and –
loaded LNA micelles were all fitted to the non-compartment
model. CUR-LNA micelles could distinctly chang pharmacokinetic
property of CUR (Table 2). LNA micelles prolonged CUR retention in
blood circulation in comparison to CUR control solution (mean
retention time, MRT0–1, 0.695 vs. 0.199 h). The half-life (t1/2z), and
The in vitro cytotoxic activity of micelles against Hela and A-549
was investigated by MTT method. After treated with blank micelles
for 48 h, the two cells’ viability was above 90% throughout the
whole range of concentrations examined during the treatment
(Fig. 10). These results showed that the CUR-free micelles did not
Fig. 10. In vitro cell cytotoxicities of CUR, blank micelles, CUR micelles, blank LNA micelles and CUR-LNA micelles against Hela and A-549cell lines (data are presented as
mean ꢁ SD (n = 3).

320
Z. Song et al. / International Journal of Pharmaceutics 471 (2014) 312–321
release and lower cytotoxicity of CUR. The particular structure of
LNA-PEG-PCL would help to efficiently encapsulate hydrophobic
drug and then change its properties in vitro or in vivo. Therefore,
CUR-LNA micelles are used as an intravenously injectable aqueous
formulation of CUR for anticancer application.
Conflict of interest
The authors report no conflicts of interest in this work.
Acknowledgments
This work is supported by the Excellent Young and Middle-aged
Scientist Award Fund of Shandong Province, China (BS2011CL006)
and Scientific Research Fund of University of Jinan (XKY1208).
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