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Imprinted-like biopolymeric micelles as efficient nanovehicles for curcumin delivery
Accepted Manuscript Title: Imprinted-like biopolymeric micelles as efficient nanovehicles for curcumin delivery Author: Lili Zhang Zeyou Qi Qiyu Huang Ke Zeng Xiaoyi Sun Juan Li You-Nian Liu PII: DOI: Reference:
S0927-7765(14)00456-1 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.08.033 COLSUB 6598
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
9-4-2014 18-8-2014 26-8-2014
Please cite this article as: L. Zhang, Z. Qi, Q. Huang, K. Zeng, X. Sun, J. Li, Y.-N. Liu, Imprinted-like biopolymeric micelles as efficient nanovehicles for curcumin delivery, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.08.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Imprinted-like biopolymeric micelles as efficient nanovehicles for
2
curcumin delivery
3
Lili Zhanga, Zeyou Qib, Qiyu Huanga, Ke Zenga, Xiaoyi Suna, Juan Lia,*, You-Nian
5
Liua,∗
cr
Department of Anesthesiology, Second Xiang-Ya Hospital, Central South
us
b
University, Changsha, Hunan 410083, China.
an
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Changsha, Hunan 410083, China.
M
8
College of Chemistry and Chemical Engineering, Central South University,
d
7
a
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∗
Corresponding authors: [email protected] (J. Li); [email protected] (Y. –N. Liu) Tel(Fax): 86‐731‐8883 6964 1
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Abstract
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To enhance the solubility and improve the bioavailability of hydrophobic curcumin, a
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new kind of imprinted-like biopolymeric micelles (IBMs) was designed. The IBMs
5
were prepared via co-assembly of gelatin-dextran conjugates with hydrophilic tea
6
polyphenol, then crosslinking the assembled micelles and finally removing the
7
template tea polyphenol by dialysis. The obtained IBMs show selective binding for
8
polyphenol analogous drugs over other drugs. Furthermore, curcumin can be
9
effectively encapsulated into the IBMs with 5×104-fold enhancement of aqueous
10
solubility. We observed the sustained drug release behaviour from the
11
curcumin-loaded IBMs (CUR@IBMs) in typical biological buffers. In addition, we
12
found the cell uptake of CUR@IBMs is much higher than that of free curcumin. The
13
cell cytotoxicity results illustrated that CUR@IBMs can improve the growth
14
inhibition of HeLa cells compared with free curcumin, while the blank IBMs have
15
little cytotoxicity. The in vivo animal study demonstrated that the IBMs could
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significantly improve the oral bioavailability of curcumin.
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Keywords
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Curcumin; Molecular imprinting; Micelle; Solubility; Oral bioavailability; Drug delivery
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1. Introduction
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Natural polyphenols such as catechins, curcumin, resveratrol, have been extensively
4
used as drugs for their anti-oxidant, anti-inflammatory and anti-cancer properties.
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Curcumin, a hydrophobic polyphenol extracted from the turmeric, exhibits
6
pharmacological effects on some diseases including cancers, Alzheimer’s,
7
cardiovascular, diabetes and so on[1-4]. As a promising anti-cancer drug, curcumin
8
could suppress carcinogenesis in various processes by activating apoptosis signaling
9
and inhibiting cell proliferation. For example, curcumin can induce apoptosis in
10
HepG-2 cells via a direct effect on mitochondria because curcumin can increase ROS
11
formation and lipid peroxidation in the cells[5]. In addition, several animal models
12
and human studies have proved that curcumin is extremely safe with low toxicity in
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vivo even at high doses[6, 7].
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On the other hand, the bioavailability of curcumin is very poor because its
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solubility in pure water is quite low (ca. 11 ng mL-1)[8], and it would suffer poor
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absorption, rapid degradation and excretion once administered in human[9, 10].
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Various techniques of pharmaceutical nanoformulations, including micelles[11],
18
nanoparticles[12] and liposomes[13], have been utilized to meet the challenges. For
19
example, Tsai et al. used poly(lactide-co-glycolide) (PLGA) nanoformulation to
20
encapsulate curcumin and found the oral bioavailability was significantly increased
21
from an animal model study[14]. Molecular imprinting technique (MIT) is known as a facile approach to create
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tailor-made binding sites with memory of the structural shape, functional groups and
2
chemical interaction of the template molecules in the highly crosslinked polymer
3
matrices, which can selectively rebind the template molecules or their analogs[15, 16].
4
Recently, MIT has attracted considerable interests in drug delivery because the
5
nanomaterials with molecular recognition can offer higher loading capacity and
6
selectivity, greater stability, lower cost and better engineering possibilities[17, 18].
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For example, Rostamizadeh et al. utilized acrylic acid and methacrylic acid to
8
fabricate the nanoparticles of molecular imprinted polymers (MIPs), which had high
9
loading capacity and achieved controlled release for naltrexone[19]. To the best of our
10
knowledge, a few imprinted nanoparticles prepared from biopolymers for drug
11
delivery have been reported. Imprinted nanoparticles based on silica nanoparticles
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coated by gelatin for drug delivery[20], and silica nanoparticles coated by chitosan for
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molecule recognition[21] have been reported recently.
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Gelatin is a widely used natural polymer for pharmaceutical and medical
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applications due to its great biodegradability and biocompatibility in physiological
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environments[22-24]. Dextran has also been employed to modify drug carriers as to
17
primarily increase the stability and the longevity of therapeutic agents in the
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circulation[25]. Previously, we prepared a new kind of complex coacervation core
19
micelles (C3Ms) by co-assembling tea polyphenol (TPP) with gelatin-dextran
20
conjugates based on hydrogen bond and hydrophobic interaction[26]. Due to the low
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cost of TPP, facile crosslinking of gelatin, high loading amount and fast complete
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release of TPP at neutral pH from the C3Ms, it is of great interest to construct the 4
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imprinted-like micelles after crosslinking and selectively removing the template TPP.
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In the present study, we introduced a new kind of imprinted-like biopolymeric
3
micelles (IBMs) for curcumin delivery. In definition, the imprinted micelle captures
4
the original template only; while imprinted-like micelle captures the template as well
5
as the template-like molecules. The IBMs were fabricated via co-assembly of
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gelatin-dextran conjugates and TPP, followed by immobilization of TPP in the cores
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through crosslinking gelatin with genipin, then removing the templated TPP from the
8
cores. The hydrophobic polyphenols can be stabilized in the cores of the IBMs with
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narrow size distribution and without macro-aggregation, different from those indirect
10
complexation with gelatin-dextran conjugates. Herein, curcumin was used as the
11
hydrophobic polyphenol model, and the loading properties, the release behavior, cell
12
uptake and cytotoxicity along with plasma pharmacokinetics of curcumin-loaded
13
IBMs (CUR@IBMs) after oral administration were investigated.
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2. Materials and methods
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2.1. Materials
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Curcumin (CUR, >98.0%) was supplied by Aladdin Chemistry Co. Ltd. (Shanghai,
17
China).
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased
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from Sigma-Aldrich. Dextran of 70 kDa was purchased from Pharmacia AB (Uppsala,
20
Sweden). Tea polyphenol (TPP, total polyphenolic content >98.0%) was supplied by
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Baishun
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(-)-Epigallocatechin-3-gallate (EGCG, >99%) was obtained from Biopurify
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Gelatin
Chemical
(type
B,
Technology
~225
Co.
Ltd.
bloom),
(Beijing,
and
China).
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Phytochemicals Co. Ltd. (Chengdu, China). Resveratrol (RES, >98%) was purchased
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from Rokey Co. Ltd. (Changsha, China). Doxorubicin hydrochloride (DOX, 99%)
3
was obtained from Huafeng Co. Ltd. (Beijing, China). Tamoxifen (TAM, >99%) and
4
indomethacin (IND, >99%) were purchased from Yuancheng Technology
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Development Co. Ltd. (Wuhan, China). Ibuprofen (IBU, >98%) was supplied by
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Shunqiang Biotechnology Co. Ltd. (Shanghai, China). 5-Fluorouracil (5-FLU, >98%)
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was purchased from Bio Basic (Toronto, Canada). Genipin (>98.0%) was purchased
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from Kangbang Biotechnology (Chengdu, China). All solutions were prepared using
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deionized water purified by a Milli-Q Water Purification System (Millipore, Bedford,
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MA, USA) to a resistance of 18.2 MΩ cm.
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2.2. Synthesis of gelatin-dextran conjugates
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Gelatin-dextran conjugates were synthesized by Maillard reaction according to
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literature procedures[27, 28]. First, gelatin and dextran with weight ratio of 1:1 were
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dissolved together in deionized water. The pH of the mixture was adjusted to 8.5
15
using 0.1 M NaOH, then the solution was lyophilized. The lyophilized power reacted
16
at 60 °C under 79% relative humidity in a desiccator containing saturated KBr
17
solution for 24 h. The Maillard products, i.e. gelatin-dextran conjugates were kept at 4
18
°C before use.
19
2.3. Preparation of IBMs
20
IBMs were prepared through a facile MIT route by using TPP as templates. Firstly,
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C3Ms were prepared by mixing TPP and gelatin-dextran conjugates as described
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previously, i.e., the C3Ms were prepared at 1:1 (w/w) of TPP to gelatin at pH 5.0, and
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the final gelatin concentration was 1.0 mg mL-1[26]. Then, the C3Ms were
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crosslinked by incubation with 10 mM genipin under stirring for 48 h at 37 oC. Finally,
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the template molecules TPP were removed by dialysis against water to produce the
4
IBMs.
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2.4. Molecular recognition properties of IBMs for polyphenols
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In order to investigate the recognition properties of the IBMs, polyphenol analogs
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including CUR, EGCG and RES, and non-polyphenol drugs including DOX, IBU,
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IND, TAM and 5-FLU were loaded into the IBMs and non-imprinted gelatin-dextran
9
conjugates, respectively. The concentration of each drug mentioned above was 0.2 mg
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mL-1 at every 1.0 mg mL-1 of gelatin in the IBMs or gelatin-dextran conjugates
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solutions. The drug binding capacities of the IBMs and conjugates (QIBMs and
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Qconjugates, mg/g) were calculated based on the amount of drug bound into the IBMs or
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conjugates according to Equation (1):
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For IBMs, QIBMs (mg/g) = Wbound / WIBMs
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For conjugates, Qconjugates (mg/g) = Wbound / Wconjugates
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(1)
where, Wbound : the amounts of bound drug;
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WIBMs : the amounts of IBMs;
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and Wconjugates : the amounts of conjugates.
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The amount of specific binding (ΔQ) by TPP-imprinted sites of IBMs to the
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analogous and non-analogous drugs was estimated by calculating the difference of
21
average binding amounts between the IBMs and the non-imprinted gelatin-dextran
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conjugates according to Equation (2): 7
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ΔQ(mg/g) = QIBMs - Qconjugates
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(2)
2.5. Preparation of CUR@IBMs
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Curcumin was loaded into the IBMs by simply mixing curcumin with the IBMs
4
solution. Briefly, curcumin was dissolved in ethanol at 10 mg mL-1, then different
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volumes of the curcumin solution were added into 1.0 mg mL-1 IBMs solution with
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various weight ratios (0.02, 0.05, 0.08, 0.10, 0.15, 0.20, 0.50, 1.00) of curcumin to
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IBMs while stirring constantly. In this process, curcumin molecules went into the
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cores of the micelles to form CUR@IBMs.
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2.6. Dynamic light scattering (DLS)
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The size distributions of the IBMs, CUR@IBMs and the redispersion of lyophilized
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CUR@IBMs were carried out on a Malvern Zetasizer Nano ZS equipped with a 4
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mW He-Ne laser (633 nm). The measurements were performed at 25 °C and a fixed
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scattering angle of 173°. The average apparent hydrodynamic diameter (Dh) and
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polydispersity index (PDI) were obtained. ζ-Potentials were also measured by
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Zetasizer Nano ZS. Each sample was analyzed at least three times.
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2.7. Morphology
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Morphological observations of the IBMs and CUR@IBMs were performed by TEM
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on a Microscope JEM-2100F at an accelerating voltage of 200 kV. Samples were
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prepared by depositing 0.5 mg mL-1 of the IBMs and CUR@IBMs solutions onto a
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carbon-coated copper grid, followed by removal of excess solution by blotting the
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grid with filter paper. The samples were dried for 72 h at room temperature in a
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desiccator containing dried silica gel. After that, the samples were negatively stained
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by phosphotungstic acid and dried for another 72 h before examination.
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2.8. Determination of drug loading capacity and encapsulation efficiency of
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CUR@IBMs
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The concentrations of curcumin were measured by absorbance at 425 nm (UV-2450,
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Shimadzu) according to the working curve obtained using standard curcumin
7
solutions. To determine the loading capacity and encapsulation efficiency of the
8
CUR@IBMs, the unloaded curcumin was separated from the CUR@IBMs by
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ultrafiltration (molecular weight cut-off of 3000 Da; Merck Millipore Ltd.). All
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experiments were performed in triplicate. The drug loading content (LC) and
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encapsulation efficiency (EE) were calculated by Equations (3) and (4), respectively:
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LC (%, w/w) = (curcumin in feed – free curcumin)/IBMs in feed×100
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EE (%, w/w) = (curcumin in feed – free curcumin)/curcumin in feed×100
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(3) (4)
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2.9. In vitro release
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The release property of CUR@IBMs was assessed by dialysis. Free curcumin solution
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with the same concentration was adopted as control. Solutions of 2.5 mL
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CUR@IBMs or free curcumin with curcumin concentration of 0.2 mg mL-1 were
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transferred into dialysis bags (cut-off molecular weight of 3500 Da) and dialyzed
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against 100 mL release medium at different pH values (0.05 M glycine-hydrochloric
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acid buffer at pH 2.0, 0.05 M phosphate buffer at pH 5.0 and 0.05 M phosphate buffer
21
at pH 6.8). 0.5% SDS was added into all the buffers in order to increase the solubility
22
of released curcumin. Then the release medium was continuously stirred for 72 h at 9
Page 9 of 33
37 °C. At predetermined sampling time, 3.0 mL of release medium was drawn out and
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replenished with 3.0 mL fresh buffer. All the samples were protected from light. The
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amount of released curcumin was measured by the UV-visible method (absorbance
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425 nm) as described above. All release experiments were performed in triplicate.
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2.10. In vitro cytotoxicity assay
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The tumor cells model, HeLa cell line, and normal cells model, L-929 mouse
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fibroblast cell line were obtained from the cell bank of Xiangya Central Laboratory of
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Central South University (Changsha, China). HeLa cells were cultured in DMEM
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with 15% fetal bovine serum, 1% penicillin and 1% streptomycin, and L-929 cells
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were cultured in RPMI-1640 with 10% fetal bovine serum, 1% penicillin and 1%
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streptomycin, respectively. The cells were seeded in 96-well plates at a density of
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5×103 cells per well and cultured in humidified environment at 37 °C containing 5%
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CO2. After 20 h when most cells anchored on to the wells, they were treated with a
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series of equivalent concentration of IBMs, free curcumin and CUR@IBMs,
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respectively. After incubation for another 48 h, 200 μL of 0.5 mg mL-1 MTT solution
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was added to each well and the plate was incubated for 4 h. After the medium was
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removed, the MTT-formazan crystals formed by the metabolically viable cells were
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dissolved in 200 μL DMSO. Finally, the absorbance of each well was measured by a
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microplate reader (Bio-TekELx800) at the wavelength of 490 nm. The cell viability
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was expressed by Equation (5):
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Cell viability (%) =AT/A0 ×100
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(5)
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Where AT is the absorbance of treated cells and A0 is the control absorbance. Data of
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cell viability are given as mean ± standard deviation (S.D.). In addition, data of cell
3
viability for each concentration are statistically compared between free curcumin and
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the CUR@IBMs using the Student’s t-test in Microsoft Excel 2007. The level of
5
significance used is p < 0.05.
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2.11. Cell uptake
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Fluorescence microscopy was used to detect the uptake of CUR@IBMs by HeLa cells
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and L-929 cells. The free curcumin solution with same concentration was adopted as
9
control. HeLa cells and L-929 cells were seeded in 24-well plates at a density of
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2.5×104 cells per well. After incubation overnight in a humidified atmosphere with
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5% CO2 at 37 °C, cells were exposed to medium containing free curcumin or
12
CUR@IBMs (the curcumin concentration was 0.02 mg mL-1). After 2 h incubation,
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the cells were washed three times with PBS at 37 oC. Then, the cells were examined at
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an exposure time of 1 s under a Nikon ECLIPSE TE 2000-U fluorescence microscope
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(20 × objective) equipped with a digital camera and a blue filter (420–490 nm
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excitation, 490–560 nm emission). Images were captured with NIS Elements F 3.0
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software (Nikon).
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2.12. In vivo oral administration
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Male Sprague-Dawley rats (200 ± 10 g body weight) were obtained from the Hunan
20
SJA Laboratory Animal Co., Ltd. (Changsha, China). All animal experiments were
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performed according to the guidelines, principles and procedures of the Xiangya
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Laboratory Animal Center of Central South University (Changsha, China) for the care
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and use of laboratory animals. Experimental rat was initially anesthetized by injecting
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0.5 mL 10% chloral hydrate. The rats were divided into two groups, group 1 (n = 6),
3
received free curcumin dispersed in water with 10% ethanol mixture; group 2 (n = 6),
4
received CUR@IBMs nanosuspension. Group 1 and 2 were fed by gavages at
5
curcumin doses of 500 mg/kg and 100 mg/kg, respectively. After oral administration,
6
400 μL blood samples were collected from the jugular vein into the EDTA-K2 tubes
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and replenished with 400 μL heparin saline at 0.5, 1.0, 1.5, 2.0, 2.5, 3.5, 4.5, 5.5 and
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6.5 h. Then, the plasma was separated by centrifuging the blood sample at 4 000 rpm
9
for 20 min. Two hundred microliter 99% ethanol was added to 200 μL of plasma for
10
protein precipitation. The samples were vortexed and centrifuged at 10 000 rpm for 30
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min. Then, 200 μL of supernatants were collected and analyzed on a fluorescence
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spectrophotometer (Hitach, F-4600, Japan) equipped with a FL Solutions Program.
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The concentration of curcumin was calculated from the standard curve of curcumin
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dissolved in ethanol/H2O 50:50 mixture. The excitation wavelength was set at 420 nm
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and the emission wavelength was found at 520 nm in all experiments.
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3. Results and discussion
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3.1. Fabrication of IBMs
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The imprinted-like biopolymeric micelles (IBMs) were prepared through a MIT-based
20
process, which creates template-shaped cavities in matrices with memory of the
21
template molecule (Scheme 1). Previously, we fabricated well-dispersed C3Ms by
22
simply mixing gelatin-dextran conjugates with TPP. The formation of C3Ms is driven 12
Page 12 of 33
by hydrophobic interaction and hydrogen bonding between the gelatin-dextran
2
conjugate and TPP. The C3Ms showed core-shell structures with high encapsulation
3
efficiency and loading capacity for TPP. Notably, the C3Ms release TPP very fast and
4
their structures are disrupted after TPP release. Otherwise, if the C3Ms are
5
crosslinked, the whole structure would not be broken after TPP removal from the
6
C3Ms. Hence, we selected TPP as the template molecule to construct a new kind of
7
biopolymeric micelles with imprinted-like properties for polyphenol drugs.
8
Furthermore, TPP has the advantages of low cost and readily removal by dialysis
9
against water.
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Genipin, as a naturally occurring reagent extracted from the fruit of Gardenia
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Jasminoides, was found to be a potential cross-linker for amino group-containing
12
biocomponents with about 5000-10000 times less cytotoxic than glutaraldehyde[29].
13
It has been used to crosslink biological tissues as well as biomaterials such as
14
gelatin-based gels, films and nanoparticles[30, 31], though the crosslinking
15
mechanisms remain unclear[29, 32]. Here, we adopted genipin as a crosslinker for the
16
fixation of C3Ms. Subsequently, the IBMs were obtained after removing TPP from
17
the crosslinked C3Ms by dialysis.
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As shown in TEM image in Fig. 1A, the IBMs show core-shell micellar structures
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with a narrow size distribution. The diameter for the micelles determined by TEM is
20
about 95 nm, smaller than the Dh of IBMs measured by DLS (ca. 170 nm). The
21
difference is probably due to the dry state of the samples in TEM observation. We
22
supposed the core is composed of gelatin with dark colour and the shell is composed 13
Page 13 of 33
of the conjugated dextran segments with light colour. Meanwhile, some
2
non-core-shell particles were also found in the Fig. 1A, which might be due to the
3
heterogeneity of staining techniques.
4
3.2. Molecular recognition of IBMs to polyphenol analogs
5
Molecular recognition of IBMs was investigated by testing the binding capacities of
6
the TPP-imprinted IBMs to polyphenol analogous drugs, i.e., CUR, EGCG and RES,
7
and to non-analogous drugs including DOX, IBU, IND, TAM and 5-FLU,
8
respectively. The IBMs exhibit higher loading capacity to polyphenol drugs than to
9
those of non-polyphenol drugs (Fig. 2A).As expected, the polyphenol analogs are able
10
to fit into the TPP-imprinted cavities due to the similarity of molecular structure and
11
the binding affinity with imprinted cavities of TPP. Besides, the non-imprinted
12
gelatin-dextran conjugates show less binding capacity to polyphenol analogous drugs
13
compared with the IBMs. The amount of specific binding (ΔQ) of TPP-imprinted
14
IBMs to the analogs is higher than that to the non-analogous polyphenol drugs (Fig.
15
2B). Also, we found the error bar in Fig. 2B is much larger than the ΔQ itself for
16
several compounds, so statistical analysis was further performed. It showed
17
significant differences between the IBMs and non-imprinted gelatin-dextran
18
conjugates for the binding of curcumin and EGCG (Fig. 2A), indicating that the IBMs
19
exhibit high selectivity for curcumin and EGCG.
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Herein, we applied the phrase “imprinted-like” to describe the molecular
21
recognition of IBMs. The consideration is that the structure of IBMs is loosely fixed
22
by crosslinked gelatin which has fewer active reaction sites than the conventional 14
Page 14 of 33
acrylic polymers, although the preparation procedure of the IBMs was similar to that
2
of the conventional imprinted polymers. We found the IBMs can recognize molecules,
3
such as EGCG and curcumin, with multiple hydroxyl groups on the aromatic rings,
4
which complies with the molecular model for the interaction of proline-rich proteins
5
(PRPs) and polyphenols[33]. The chemical structure for the polyphenols contains
6
several phenolic groups. The structure of PRPs (e.g. gelatin) is rich in proline residue
7
and requires an extended conformation. The binding of polyphenols to PRPs is mainly
8
based on hydrogen bonding and hydrophobic interaction between hydrophobic amino
9
acid residues, mostly proline, and phenol rings of polyphenols.
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3.3. Loading curcumin into IBMs
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As shown in Fig. 3, free curcumin can hardly dissolve or disperse in the aqueous
12
media without additives. While, curcumin can disperse in the solution of
13
gelatin-dextran conjugates in the beginning, but within hours the precipitation
14
occurred. By contrast, curcumin was well dispersed after it was encapsulated into the
15
IBMs (Fig. 3 and Table S1). In particular, the CUR@IBMs can be redispersed after
16
lyophilisation without any excipient (Fig. 3).
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Presently, the micelles used as carriers for curcumin are mostly composed of
18
amphiphilic block polymers in which curcumin could be well dispersed in the
19
hydrophobic regions[34, 35]. However, how to stabilize curcumin in hydrophilic
20
polymers is still a big challenge. As mentioned above, the highly hydrophilic
21
gelatin-dextran conjugates couldn’t well stabilize the hydrophobic curcumin.
22
Alternatively, the IBMs we constructed could effectively encapsulate curcumin, 15
Page 15 of 33
1
which is able to disperse in the aqueous solution. The high stability of the
2
CUR@IBMs is ascribed to the cavities of IBMs which is imprinted by TPP. The EE of the CUR@IBMs is as much as 99% and remains unchangeable until the
4
concentration of curcumin is above 0.5 mg mL−1 (Fig. S1). The LC has a linear
5
relationship with curcumin concentration (R2 = 0.99), suggesting the loading amount
6
could be well controlled by adjusting the concentration of curcumin in feed. The
7
aqueous solubility of curcumin could be largely enhanced from the original 11 ng
8
mL-1 to 0.5 mg mL-1 after encapsulation into the IBMs, about 5×104-fold increase.
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As shown in Fig. 1B, the CUR@IBMs have spherical structures. Meanwhile, we
10
found the Dh of CUR@IBMs slightly increased with the increase of curcumin
11
concentration (Table S1). Nevertheless, the sizes (about 160 nm ~ 220 nm) are still
12
suitable for the enhanced permeability and retention (EPR) effect, accumulating high
13
local curcumin concentration in tumors.
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FT-IR spectra of CM@IBMs, free curcumin and IBMs were measured to
15
investigate the interaction between curcumin and IBMs (Fig. S2, Supplementary data).
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The characteristic absorption regions around 3400 cm-1 can be assigned to the
17
vibrational band of the hydroxyl group of IBMs. However, a band shift from 3432
18
cm-1 to 3416 cm-1 is observed in the spectrum of CUR@IBMs powder compared with
19
that of IBMs, attributing to the formation of intermolecular hydrogen bonds between
20
the IBMs and curcumin[36].The result is in agreement with the formation mechanism
21
of co-assembly of C3Ms, which is mainly driven by hydrogen bonding and
22
hydrophobic interaction[26]. 16
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3.4. In vitro drug release
2
It has been reported that free curcumin would be quickly eliminated in the metabolic
3
process[37]. Nano-drug delivery systems may provide an alternative way to protect
4
curcumin from the metabolism before curcumin arrives the target sites. In this respect,
5
sustained release of curcumin is required. The release profiles of CUR@IBMs were
6
obtained in three media at pH 2.0, pH 5.0 and pH 6.8, simulating various
7
physiological environments. Three different pH values were selected in order to
8
simulate various physiological environments, including stomach (pH 1.0 ~ 2.5),
9
intestine (pH 6.4 ~ 7.5), blood and normal tissue (pH 7.4 ~ 7.5), intracellular
10
components (pH ranging from 4.5 in the lysosome to 8.0 in the mitochondria), and
11
solid tumor extracellular microenvironment (pH < 6.5)[38, 39]. As illustrated in Fig. 4,
12
curcumin was released much slower after being encapsulated by the IBMs compared
13
with free curcumin, which reveals sustained release of CUR@IBMs. For example,
14
after 72 h, 54%, 47% and 60% of the curcumin were released from the IBMs at pH
15
2.0, pH 5.0 and pH 6.8, respectively; by contrast, free curcumin had completed its
16
release at the three pHs. In addition, the CUR@IBMs themselves are highly
17
dispersible at the three pHs studied. The reason for the pH influence on the release of
18
IBMs may be due to the affinity of IBMs and curcumin. Previously, we have found
19
that polyphenols exhibit the strongest binding with the C3Ms at the isoelectric point
20
(pI) of gelatin (pI ≈ 5.0)[26], which could explain the least release of curcumin (47%)
21
from IBMs. While, the affinity would be weakened at pH 2.0 and pH 6.8, and led to
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more curcumin release from the cores of the IBMs, about 54% and 60%, respectively.
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It suggests that the CUR@IBMs can be used as sustained formulations applied either
2
in oral administration or in intravenous administration.
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3.5. Cell cytotoxicity
4
The cell cytotoxicities of free curcumin, IBMs and CUR@IBMs were evaluated on
5
HeLa cells using MTT assay. The results revealed that both free curcumin and
6
CUR@IBMs display a concentration-dependent cytotoxicity against the HeLa cells,
7
while the IBMs show little cytotoxicity at the equivalent concentrations to the
8
CUR@IBMs. The encapsulated curcumin presents comparative or better cytotoxic
9
effects than free curcumin (see Fig. 5A).
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In addition, the IBMs show little cytotoxicity at the equivalent concentrations to the
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CUR@IBMs towards normal L-929 cells (Fig. 5B). Meanwhile, CUR@IBMs showed
12
less cytotoxicity on the L-929 cells than on the HeLa cells. The result is in agreement
13
with other cell cytotoxicity studies of curcumin encapsulated by PLGA-MNPs and
14
N,O-carboxymethyl chitosan nanoparticles[40, 41], which may be due to the healthy
15
nature of the normal cells compared with the cancer cells.
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To further confirm the relationship between the anti-cancer activities and the cell
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uptake, we observed the fluorescence of HeLa cells after incubation with free
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curcumin and CUR@IBMs, respectively. The fluorescent images show that the
19
fluorescence of cells treated with free curcumin is relatively faint (Fig. 6). Whereas,
20
the fluorescence of HeLa cells is greatly enhanced, and it can be clearly observed in
21
the cytoplasm of HeLa cells after treated with CUR@IBMs. Our results are consistent
22
with other curcumin nanoparticulate formulations which were more effective than 18
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native curcumin against the growth of cancer cell lines due to the enhanced cell
2
uptake[42, 43]. In addition, L-929 cells incubated with CUR@IBMs showed faint
3
fluorescence intensity (Fig. 6), which implies that the less cell uptake of curcumin for
4
L-929 cells than that for HeLa cells, confirming the less cytotoxicity for L-929 cells.
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3.6. In vivo oral bioavailability
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To understand the relative bioavailability of CUR@IBMs, plasma pharmacokinetic
7
after oral administration was studied on rat model by using free curcumin suspension
8
as control. The concentration of curcumin was monitored by fluorescence method.
9
Curcumin gives fluorescence emission at 520 nm when it is excited at 420 nm, which
10
has attracted many researches on fluorescent property of curcumin as a potential
11
fluorescent probe and photodynamic therapy drug[44, 45]. Fig. 7A shows the
12
fluorescence emission spectra of the detected curcumin in rat plasma samples after
13
oral administration for 1.5 h. Notably, the curcumin concentrations in rat plasma
14
exhibit significant increases following oral administration of 100 mg/kg CUR@IBMs
15
nanoparticles compared with 500 mg/kg free curcumin (Fig. 7A and 7B). In the
16
plasma of untreated rat, the fluorescence was negligible under the same excitation.
17
The results demonstrated the relative bioavailability of CUR@IBMs is higher than
18
that of free curcumin. Similar phenomena were observed in other curcumin
19
nanoformulation[14, 46]. As mentioned above, the bioavailability of curcumin is
20
limited by its low solubility, poor absorption, rapid degradation and excretion[3, 9].
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The significant improvement of bioavailability in oral administration may be mainly
22
attributed to the higher absorption from the enhancement of solubility. Additionally,
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the IBMs could protect curcumin against metabolism in the gastrointestinal tract and
2
in blood circulation before curcumin is released from nanoparticles, which could be
3
another reason attributed to the improvement of bioavailability.
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4. Conclusion
6
The novel IBMs were successfully prepared by a simple and green process and they
7
can specifically bind to polyphenol analogous drugs. High encapsulation of curcumin
8
in the IBMs facilitates complete dispersion of curcumin in water. The CUR@IBMs
9
possess the advantages of narrow size distribution, high drug encapsulation efficiency
10
and sustained release. Meanwhile, they display enhanced cell uptake, superior growth
11
inhibition against tumor cells and improved oral bioavailability over free curcumin,
12
indicating that the IBMs are potential nanovehicles for curcumin delivery in cancer
13
therapy.
14
Acknowledgements
15
This work was financially supported by the National Natural Science Foundation of
16
China (Nos. 21374133, 21276285 and 21104096), Hunan Provincial Natural Science
17
Foundation of China (No. 12JJ4014), China Postdoctoral Science Foundation (No.
18
2011M501281) and Postgraduate Science Foundation of Central South University
19
(No. 2013zzts161).
20
Appendix A. Supplementary data
21
Supplementary material related to this article can be found in the online version, at
22
http://dx.doi.org/10.1016/
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Fig. 2. (A) Binding capacity (Q) of the polyphenol analogous and non-analogous drugs in the IBMs and non-imprinted gelatin-dextran conjugates. (B) The specific binding (ΔQ) by the IBMs to polyphenol analogous and non-analogous drugs. Data of Q for each drug are statistically compared between IBMs and the conjugates using Student’s t-test in Microsoft Excel 2003. The level of significance is p < 0.05. Asterisk means a significant difference. Error bars for ΔQ represent the standard error (n = 3). Fig. 3. Size distributions of CUR@IBMs as-prepared and after lyophilization and then rehydration analyzed by DLS. Inset: Photos of free curcumin in water (left); curcumin and gelatin-dextran conjugates mixture (middle); CUR@IBMs (right). Fig. 4. Curcumin release profiles against different release medium buffers at pH 2.0 (A), pH 5.0 (B) and pH 6.8 (C). Fig. 5. Cell cytotoxicities of the IBMs, free curcumin and CUR@IBMs against HeLa cells (A) and 22
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L-929 cells (B) after 48 h incubation. The concentrations of IBMs are equivalent to those of CUR@IBMs. Asterisk means a significant difference (*p< 0.05).
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Scheme 1. Preparation of IBMs and CUR@IBMs.
Fig. 6. Bright and fluorescent microscopic images for the HeLa cell and L-929 cell uptake of free curcumin and CUR@IBMs after incubation for 2 h, respectively.
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Fig. 7. (A) The fluorescence emission spectra of CUR@IBMs in plasma (solid), free curcumin in plasma (dot), and untreated plasma (dash) after oral administration in rats for 1.5 h. Inset: The fluorescence emission spectra of free curcumin. (B) Concentration curve of curcumin in rat plasma after oral administration with free curcumin (500 mg/kg) and CUR@IBMs (100 mg/kg).
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Graphical Abstract
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Imprinted-like biopolymeric micelles (IBMs) for hydrophobic curcumin delivery were developed by using gelatin-dextran conjugate as an imprinting material and tea polyphenol (TPP) as a template.
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Highlights We prepared imprinted-like biopolymeric micelles (IBMs) via green process; IBMs showed imprinted-like property for the polyphenol analogous drugs; Water solubility of curcumin was increased by 5×104-fold after encapsulation; Curcumin-loaded IBMs exhibited superior growth inhibition against tumor cells; IBMs could effectively improve the oral bioavailability of curcumin.
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S0927-7765(14)00456-1 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.08.033 COLSUB 6598
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
9-4-2014 18-8-2014 26-8-2014
Please cite this article as: L. Zhang, Z. Qi, Q. Huang, K. Zeng, X. Sun, J. Li, Y.-N. Liu, Imprinted-like biopolymeric micelles as efficient nanovehicles for curcumin delivery, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.08.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Imprinted-like biopolymeric micelles as efficient nanovehicles for
2
curcumin delivery
3
Lili Zhanga, Zeyou Qib, Qiyu Huanga, Ke Zenga, Xiaoyi Suna, Juan Lia,*, You-Nian
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Liua,∗
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Department of Anesthesiology, Second Xiang-Ya Hospital, Central South
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b
University, Changsha, Hunan 410083, China.
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Changsha, Hunan 410083, China.
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College of Chemistry and Chemical Engineering, Central South University,
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∗
Corresponding authors: [email protected] (J. Li); [email protected] (Y. –N. Liu) Tel(Fax): 86‐731‐8883 6964 1
Page 1 of 33
1
Abstract
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To enhance the solubility and improve the bioavailability of hydrophobic curcumin, a
4
new kind of imprinted-like biopolymeric micelles (IBMs) was designed. The IBMs
5
were prepared via co-assembly of gelatin-dextran conjugates with hydrophilic tea
6
polyphenol, then crosslinking the assembled micelles and finally removing the
7
template tea polyphenol by dialysis. The obtained IBMs show selective binding for
8
polyphenol analogous drugs over other drugs. Furthermore, curcumin can be
9
effectively encapsulated into the IBMs with 5×104-fold enhancement of aqueous
10
solubility. We observed the sustained drug release behaviour from the
11
curcumin-loaded IBMs (CUR@IBMs) in typical biological buffers. In addition, we
12
found the cell uptake of CUR@IBMs is much higher than that of free curcumin. The
13
cell cytotoxicity results illustrated that CUR@IBMs can improve the growth
14
inhibition of HeLa cells compared with free curcumin, while the blank IBMs have
15
little cytotoxicity. The in vivo animal study demonstrated that the IBMs could
16
significantly improve the oral bioavailability of curcumin.
17
Keywords
18 19
Curcumin; Molecular imprinting; Micelle; Solubility; Oral bioavailability; Drug delivery
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1. Introduction
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Natural polyphenols such as catechins, curcumin, resveratrol, have been extensively
4
used as drugs for their anti-oxidant, anti-inflammatory and anti-cancer properties.
5
Curcumin, a hydrophobic polyphenol extracted from the turmeric, exhibits
6
pharmacological effects on some diseases including cancers, Alzheimer’s,
7
cardiovascular, diabetes and so on[1-4]. As a promising anti-cancer drug, curcumin
8
could suppress carcinogenesis in various processes by activating apoptosis signaling
9
and inhibiting cell proliferation. For example, curcumin can induce apoptosis in
10
HepG-2 cells via a direct effect on mitochondria because curcumin can increase ROS
11
formation and lipid peroxidation in the cells[5]. In addition, several animal models
12
and human studies have proved that curcumin is extremely safe with low toxicity in
13
vivo even at high doses[6, 7].
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On the other hand, the bioavailability of curcumin is very poor because its
15
solubility in pure water is quite low (ca. 11 ng mL-1)[8], and it would suffer poor
16
absorption, rapid degradation and excretion once administered in human[9, 10].
17
Various techniques of pharmaceutical nanoformulations, including micelles[11],
18
nanoparticles[12] and liposomes[13], have been utilized to meet the challenges. For
19
example, Tsai et al. used poly(lactide-co-glycolide) (PLGA) nanoformulation to
20
encapsulate curcumin and found the oral bioavailability was significantly increased
21
from an animal model study[14]. Molecular imprinting technique (MIT) is known as a facile approach to create
22
3
Page 3 of 33
tailor-made binding sites with memory of the structural shape, functional groups and
2
chemical interaction of the template molecules in the highly crosslinked polymer
3
matrices, which can selectively rebind the template molecules or their analogs[15, 16].
4
Recently, MIT has attracted considerable interests in drug delivery because the
5
nanomaterials with molecular recognition can offer higher loading capacity and
6
selectivity, greater stability, lower cost and better engineering possibilities[17, 18].
7
For example, Rostamizadeh et al. utilized acrylic acid and methacrylic acid to
8
fabricate the nanoparticles of molecular imprinted polymers (MIPs), which had high
9
loading capacity and achieved controlled release for naltrexone[19]. To the best of our
10
knowledge, a few imprinted nanoparticles prepared from biopolymers for drug
11
delivery have been reported. Imprinted nanoparticles based on silica nanoparticles
12
coated by gelatin for drug delivery[20], and silica nanoparticles coated by chitosan for
13
molecule recognition[21] have been reported recently.
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Gelatin is a widely used natural polymer for pharmaceutical and medical
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applications due to its great biodegradability and biocompatibility in physiological
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environments[22-24]. Dextran has also been employed to modify drug carriers as to
17
primarily increase the stability and the longevity of therapeutic agents in the
18
circulation[25]. Previously, we prepared a new kind of complex coacervation core
19
micelles (C3Ms) by co-assembling tea polyphenol (TPP) with gelatin-dextran
20
conjugates based on hydrogen bond and hydrophobic interaction[26]. Due to the low
21
cost of TPP, facile crosslinking of gelatin, high loading amount and fast complete
22
release of TPP at neutral pH from the C3Ms, it is of great interest to construct the 4
Page 4 of 33
imprinted-like micelles after crosslinking and selectively removing the template TPP.
2
In the present study, we introduced a new kind of imprinted-like biopolymeric
3
micelles (IBMs) for curcumin delivery. In definition, the imprinted micelle captures
4
the original template only; while imprinted-like micelle captures the template as well
5
as the template-like molecules. The IBMs were fabricated via co-assembly of
6
gelatin-dextran conjugates and TPP, followed by immobilization of TPP in the cores
7
through crosslinking gelatin with genipin, then removing the templated TPP from the
8
cores. The hydrophobic polyphenols can be stabilized in the cores of the IBMs with
9
narrow size distribution and without macro-aggregation, different from those indirect
10
complexation with gelatin-dextran conjugates. Herein, curcumin was used as the
11
hydrophobic polyphenol model, and the loading properties, the release behavior, cell
12
uptake and cytotoxicity along with plasma pharmacokinetics of curcumin-loaded
13
IBMs (CUR@IBMs) after oral administration were investigated.
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2. Materials and methods
15
2.1. Materials
16
Curcumin (CUR, >98.0%) was supplied by Aladdin Chemistry Co. Ltd. (Shanghai,
17
China).
18
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased
19
from Sigma-Aldrich. Dextran of 70 kDa was purchased from Pharmacia AB (Uppsala,
20
Sweden). Tea polyphenol (TPP, total polyphenolic content >98.0%) was supplied by
21
Baishun
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(-)-Epigallocatechin-3-gallate (EGCG, >99%) was obtained from Biopurify
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Gelatin
Chemical
(type
B,
Technology
~225
Co.
Ltd.
bloom),
(Beijing,
and
China).
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Phytochemicals Co. Ltd. (Chengdu, China). Resveratrol (RES, >98%) was purchased
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from Rokey Co. Ltd. (Changsha, China). Doxorubicin hydrochloride (DOX, 99%)
3
was obtained from Huafeng Co. Ltd. (Beijing, China). Tamoxifen (TAM, >99%) and
4
indomethacin (IND, >99%) were purchased from Yuancheng Technology
5
Development Co. Ltd. (Wuhan, China). Ibuprofen (IBU, >98%) was supplied by
6
Shunqiang Biotechnology Co. Ltd. (Shanghai, China). 5-Fluorouracil (5-FLU, >98%)
7
was purchased from Bio Basic (Toronto, Canada). Genipin (>98.0%) was purchased
8
from Kangbang Biotechnology (Chengdu, China). All solutions were prepared using
9
deionized water purified by a Milli-Q Water Purification System (Millipore, Bedford,
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MA, USA) to a resistance of 18.2 MΩ cm.
11
2.2. Synthesis of gelatin-dextran conjugates
12
Gelatin-dextran conjugates were synthesized by Maillard reaction according to
13
literature procedures[27, 28]. First, gelatin and dextran with weight ratio of 1:1 were
14
dissolved together in deionized water. The pH of the mixture was adjusted to 8.5
15
using 0.1 M NaOH, then the solution was lyophilized. The lyophilized power reacted
16
at 60 °C under 79% relative humidity in a desiccator containing saturated KBr
17
solution for 24 h. The Maillard products, i.e. gelatin-dextran conjugates were kept at 4
18
°C before use.
19
2.3. Preparation of IBMs
20
IBMs were prepared through a facile MIT route by using TPP as templates. Firstly,
21
C3Ms were prepared by mixing TPP and gelatin-dextran conjugates as described
22
previously, i.e., the C3Ms were prepared at 1:1 (w/w) of TPP to gelatin at pH 5.0, and
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Page 6 of 33
the final gelatin concentration was 1.0 mg mL-1[26]. Then, the C3Ms were
2
crosslinked by incubation with 10 mM genipin under stirring for 48 h at 37 oC. Finally,
3
the template molecules TPP were removed by dialysis against water to produce the
4
IBMs.
5
2.4. Molecular recognition properties of IBMs for polyphenols
6
In order to investigate the recognition properties of the IBMs, polyphenol analogs
7
including CUR, EGCG and RES, and non-polyphenol drugs including DOX, IBU,
8
IND, TAM and 5-FLU were loaded into the IBMs and non-imprinted gelatin-dextran
9
conjugates, respectively. The concentration of each drug mentioned above was 0.2 mg
10
mL-1 at every 1.0 mg mL-1 of gelatin in the IBMs or gelatin-dextran conjugates
11
solutions. The drug binding capacities of the IBMs and conjugates (QIBMs and
12
Qconjugates, mg/g) were calculated based on the amount of drug bound into the IBMs or
13
conjugates according to Equation (1):
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For IBMs, QIBMs (mg/g) = Wbound / WIBMs
14
For conjugates, Qconjugates (mg/g) = Wbound / Wconjugates
15
(1)
where, Wbound : the amounts of bound drug;
16
WIBMs : the amounts of IBMs;
17
and Wconjugates : the amounts of conjugates.
18 19
The amount of specific binding (ΔQ) by TPP-imprinted sites of IBMs to the
20
analogous and non-analogous drugs was estimated by calculating the difference of
21
average binding amounts between the IBMs and the non-imprinted gelatin-dextran
22
conjugates according to Equation (2): 7
Page 7 of 33
ΔQ(mg/g) = QIBMs - Qconjugates
1
(2)
2.5. Preparation of CUR@IBMs
3
Curcumin was loaded into the IBMs by simply mixing curcumin with the IBMs
4
solution. Briefly, curcumin was dissolved in ethanol at 10 mg mL-1, then different
5
volumes of the curcumin solution were added into 1.0 mg mL-1 IBMs solution with
6
various weight ratios (0.02, 0.05, 0.08, 0.10, 0.15, 0.20, 0.50, 1.00) of curcumin to
7
IBMs while stirring constantly. In this process, curcumin molecules went into the
8
cores of the micelles to form CUR@IBMs.
9
2.6. Dynamic light scattering (DLS)
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The size distributions of the IBMs, CUR@IBMs and the redispersion of lyophilized
11
CUR@IBMs were carried out on a Malvern Zetasizer Nano ZS equipped with a 4
12
mW He-Ne laser (633 nm). The measurements were performed at 25 °C and a fixed
13
scattering angle of 173°. The average apparent hydrodynamic diameter (Dh) and
14
polydispersity index (PDI) were obtained. ζ-Potentials were also measured by
15
Zetasizer Nano ZS. Each sample was analyzed at least three times.
16
2.7. Morphology
17
Morphological observations of the IBMs and CUR@IBMs were performed by TEM
18
on a Microscope JEM-2100F at an accelerating voltage of 200 kV. Samples were
19
prepared by depositing 0.5 mg mL-1 of the IBMs and CUR@IBMs solutions onto a
20
carbon-coated copper grid, followed by removal of excess solution by blotting the
21
grid with filter paper. The samples were dried for 72 h at room temperature in a
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Page 8 of 33
desiccator containing dried silica gel. After that, the samples were negatively stained
2
by phosphotungstic acid and dried for another 72 h before examination.
3
2.8. Determination of drug loading capacity and encapsulation efficiency of
4
CUR@IBMs
5
The concentrations of curcumin were measured by absorbance at 425 nm (UV-2450,
6
Shimadzu) according to the working curve obtained using standard curcumin
7
solutions. To determine the loading capacity and encapsulation efficiency of the
8
CUR@IBMs, the unloaded curcumin was separated from the CUR@IBMs by
9
ultrafiltration (molecular weight cut-off of 3000 Da; Merck Millipore Ltd.). All
10
experiments were performed in triplicate. The drug loading content (LC) and
11
encapsulation efficiency (EE) were calculated by Equations (3) and (4), respectively:
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LC (%, w/w) = (curcumin in feed – free curcumin)/IBMs in feed×100
13
EE (%, w/w) = (curcumin in feed – free curcumin)/curcumin in feed×100
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(3) (4)
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2.9. In vitro release
15
The release property of CUR@IBMs was assessed by dialysis. Free curcumin solution
16
with the same concentration was adopted as control. Solutions of 2.5 mL
17
CUR@IBMs or free curcumin with curcumin concentration of 0.2 mg mL-1 were
18
transferred into dialysis bags (cut-off molecular weight of 3500 Da) and dialyzed
19
against 100 mL release medium at different pH values (0.05 M glycine-hydrochloric
20
acid buffer at pH 2.0, 0.05 M phosphate buffer at pH 5.0 and 0.05 M phosphate buffer
21
at pH 6.8). 0.5% SDS was added into all the buffers in order to increase the solubility
22
of released curcumin. Then the release medium was continuously stirred for 72 h at 9
Page 9 of 33
37 °C. At predetermined sampling time, 3.0 mL of release medium was drawn out and
2
replenished with 3.0 mL fresh buffer. All the samples were protected from light. The
3
amount of released curcumin was measured by the UV-visible method (absorbance
4
425 nm) as described above. All release experiments were performed in triplicate.
5
2.10. In vitro cytotoxicity assay
6
The tumor cells model, HeLa cell line, and normal cells model, L-929 mouse
7
fibroblast cell line were obtained from the cell bank of Xiangya Central Laboratory of
8
Central South University (Changsha, China). HeLa cells were cultured in DMEM
9
with 15% fetal bovine serum, 1% penicillin and 1% streptomycin, and L-929 cells
10
were cultured in RPMI-1640 with 10% fetal bovine serum, 1% penicillin and 1%
11
streptomycin, respectively. The cells were seeded in 96-well plates at a density of
12
5×103 cells per well and cultured in humidified environment at 37 °C containing 5%
13
CO2. After 20 h when most cells anchored on to the wells, they were treated with a
14
series of equivalent concentration of IBMs, free curcumin and CUR@IBMs,
15
respectively. After incubation for another 48 h, 200 μL of 0.5 mg mL-1 MTT solution
16
was added to each well and the plate was incubated for 4 h. After the medium was
17
removed, the MTT-formazan crystals formed by the metabolically viable cells were
18
dissolved in 200 μL DMSO. Finally, the absorbance of each well was measured by a
19
microplate reader (Bio-TekELx800) at the wavelength of 490 nm. The cell viability
20
was expressed by Equation (5):
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Cell viability (%) =AT/A0 ×100
21
(5)
10
Page 10 of 33
Where AT is the absorbance of treated cells and A0 is the control absorbance. Data of
2
cell viability are given as mean ± standard deviation (S.D.). In addition, data of cell
3
viability for each concentration are statistically compared between free curcumin and
4
the CUR@IBMs using the Student’s t-test in Microsoft Excel 2007. The level of
5
significance used is p < 0.05.
6
2.11. Cell uptake
7
Fluorescence microscopy was used to detect the uptake of CUR@IBMs by HeLa cells
8
and L-929 cells. The free curcumin solution with same concentration was adopted as
9
control. HeLa cells and L-929 cells were seeded in 24-well plates at a density of
10
2.5×104 cells per well. After incubation overnight in a humidified atmosphere with
11
5% CO2 at 37 °C, cells were exposed to medium containing free curcumin or
12
CUR@IBMs (the curcumin concentration was 0.02 mg mL-1). After 2 h incubation,
13
the cells were washed three times with PBS at 37 oC. Then, the cells were examined at
14
an exposure time of 1 s under a Nikon ECLIPSE TE 2000-U fluorescence microscope
15
(20 × objective) equipped with a digital camera and a blue filter (420–490 nm
16
excitation, 490–560 nm emission). Images were captured with NIS Elements F 3.0
17
software (Nikon).
18
2.12. In vivo oral administration
19
Male Sprague-Dawley rats (200 ± 10 g body weight) were obtained from the Hunan
20
SJA Laboratory Animal Co., Ltd. (Changsha, China). All animal experiments were
21
performed according to the guidelines, principles and procedures of the Xiangya
22
Laboratory Animal Center of Central South University (Changsha, China) for the care
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Page 11 of 33
and use of laboratory animals. Experimental rat was initially anesthetized by injecting
2
0.5 mL 10% chloral hydrate. The rats were divided into two groups, group 1 (n = 6),
3
received free curcumin dispersed in water with 10% ethanol mixture; group 2 (n = 6),
4
received CUR@IBMs nanosuspension. Group 1 and 2 were fed by gavages at
5
curcumin doses of 500 mg/kg and 100 mg/kg, respectively. After oral administration,
6
400 μL blood samples were collected from the jugular vein into the EDTA-K2 tubes
7
and replenished with 400 μL heparin saline at 0.5, 1.0, 1.5, 2.0, 2.5, 3.5, 4.5, 5.5 and
8
6.5 h. Then, the plasma was separated by centrifuging the blood sample at 4 000 rpm
9
for 20 min. Two hundred microliter 99% ethanol was added to 200 μL of plasma for
10
protein precipitation. The samples were vortexed and centrifuged at 10 000 rpm for 30
11
min. Then, 200 μL of supernatants were collected and analyzed on a fluorescence
12
spectrophotometer (Hitach, F-4600, Japan) equipped with a FL Solutions Program.
13
The concentration of curcumin was calculated from the standard curve of curcumin
14
dissolved in ethanol/H2O 50:50 mixture. The excitation wavelength was set at 420 nm
15
and the emission wavelength was found at 520 nm in all experiments.
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16 17
3. Results and discussion
18
3.1. Fabrication of IBMs
19
The imprinted-like biopolymeric micelles (IBMs) were prepared through a MIT-based
20
process, which creates template-shaped cavities in matrices with memory of the
21
template molecule (Scheme 1). Previously, we fabricated well-dispersed C3Ms by
22
simply mixing gelatin-dextran conjugates with TPP. The formation of C3Ms is driven 12
Page 12 of 33
by hydrophobic interaction and hydrogen bonding between the gelatin-dextran
2
conjugate and TPP. The C3Ms showed core-shell structures with high encapsulation
3
efficiency and loading capacity for TPP. Notably, the C3Ms release TPP very fast and
4
their structures are disrupted after TPP release. Otherwise, if the C3Ms are
5
crosslinked, the whole structure would not be broken after TPP removal from the
6
C3Ms. Hence, we selected TPP as the template molecule to construct a new kind of
7
biopolymeric micelles with imprinted-like properties for polyphenol drugs.
8
Furthermore, TPP has the advantages of low cost and readily removal by dialysis
9
against water.
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Genipin, as a naturally occurring reagent extracted from the fruit of Gardenia
11
Jasminoides, was found to be a potential cross-linker for amino group-containing
12
biocomponents with about 5000-10000 times less cytotoxic than glutaraldehyde[29].
13
It has been used to crosslink biological tissues as well as biomaterials such as
14
gelatin-based gels, films and nanoparticles[30, 31], though the crosslinking
15
mechanisms remain unclear[29, 32]. Here, we adopted genipin as a crosslinker for the
16
fixation of C3Ms. Subsequently, the IBMs were obtained after removing TPP from
17
the crosslinked C3Ms by dialysis.
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As shown in TEM image in Fig. 1A, the IBMs show core-shell micellar structures
19
with a narrow size distribution. The diameter for the micelles determined by TEM is
20
about 95 nm, smaller than the Dh of IBMs measured by DLS (ca. 170 nm). The
21
difference is probably due to the dry state of the samples in TEM observation. We
22
supposed the core is composed of gelatin with dark colour and the shell is composed 13
Page 13 of 33
of the conjugated dextran segments with light colour. Meanwhile, some
2
non-core-shell particles were also found in the Fig. 1A, which might be due to the
3
heterogeneity of staining techniques.
4
3.2. Molecular recognition of IBMs to polyphenol analogs
5
Molecular recognition of IBMs was investigated by testing the binding capacities of
6
the TPP-imprinted IBMs to polyphenol analogous drugs, i.e., CUR, EGCG and RES,
7
and to non-analogous drugs including DOX, IBU, IND, TAM and 5-FLU,
8
respectively. The IBMs exhibit higher loading capacity to polyphenol drugs than to
9
those of non-polyphenol drugs (Fig. 2A).As expected, the polyphenol analogs are able
10
to fit into the TPP-imprinted cavities due to the similarity of molecular structure and
11
the binding affinity with imprinted cavities of TPP. Besides, the non-imprinted
12
gelatin-dextran conjugates show less binding capacity to polyphenol analogous drugs
13
compared with the IBMs. The amount of specific binding (ΔQ) of TPP-imprinted
14
IBMs to the analogs is higher than that to the non-analogous polyphenol drugs (Fig.
15
2B). Also, we found the error bar in Fig. 2B is much larger than the ΔQ itself for
16
several compounds, so statistical analysis was further performed. It showed
17
significant differences between the IBMs and non-imprinted gelatin-dextran
18
conjugates for the binding of curcumin and EGCG (Fig. 2A), indicating that the IBMs
19
exhibit high selectivity for curcumin and EGCG.
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20
Herein, we applied the phrase “imprinted-like” to describe the molecular
21
recognition of IBMs. The consideration is that the structure of IBMs is loosely fixed
22
by crosslinked gelatin which has fewer active reaction sites than the conventional 14
Page 14 of 33
acrylic polymers, although the preparation procedure of the IBMs was similar to that
2
of the conventional imprinted polymers. We found the IBMs can recognize molecules,
3
such as EGCG and curcumin, with multiple hydroxyl groups on the aromatic rings,
4
which complies with the molecular model for the interaction of proline-rich proteins
5
(PRPs) and polyphenols[33]. The chemical structure for the polyphenols contains
6
several phenolic groups. The structure of PRPs (e.g. gelatin) is rich in proline residue
7
and requires an extended conformation. The binding of polyphenols to PRPs is mainly
8
based on hydrogen bonding and hydrophobic interaction between hydrophobic amino
9
acid residues, mostly proline, and phenol rings of polyphenols.
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3.3. Loading curcumin into IBMs
11
As shown in Fig. 3, free curcumin can hardly dissolve or disperse in the aqueous
12
media without additives. While, curcumin can disperse in the solution of
13
gelatin-dextran conjugates in the beginning, but within hours the precipitation
14
occurred. By contrast, curcumin was well dispersed after it was encapsulated into the
15
IBMs (Fig. 3 and Table S1). In particular, the CUR@IBMs can be redispersed after
16
lyophilisation without any excipient (Fig. 3).
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Presently, the micelles used as carriers for curcumin are mostly composed of
18
amphiphilic block polymers in which curcumin could be well dispersed in the
19
hydrophobic regions[34, 35]. However, how to stabilize curcumin in hydrophilic
20
polymers is still a big challenge. As mentioned above, the highly hydrophilic
21
gelatin-dextran conjugates couldn’t well stabilize the hydrophobic curcumin.
22
Alternatively, the IBMs we constructed could effectively encapsulate curcumin, 15
Page 15 of 33
1
which is able to disperse in the aqueous solution. The high stability of the
2
CUR@IBMs is ascribed to the cavities of IBMs which is imprinted by TPP. The EE of the CUR@IBMs is as much as 99% and remains unchangeable until the
4
concentration of curcumin is above 0.5 mg mL−1 (Fig. S1). The LC has a linear
5
relationship with curcumin concentration (R2 = 0.99), suggesting the loading amount
6
could be well controlled by adjusting the concentration of curcumin in feed. The
7
aqueous solubility of curcumin could be largely enhanced from the original 11 ng
8
mL-1 to 0.5 mg mL-1 after encapsulation into the IBMs, about 5×104-fold increase.
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As shown in Fig. 1B, the CUR@IBMs have spherical structures. Meanwhile, we
10
found the Dh of CUR@IBMs slightly increased with the increase of curcumin
11
concentration (Table S1). Nevertheless, the sizes (about 160 nm ~ 220 nm) are still
12
suitable for the enhanced permeability and retention (EPR) effect, accumulating high
13
local curcumin concentration in tumors.
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FT-IR spectra of CM@IBMs, free curcumin and IBMs were measured to
15
investigate the interaction between curcumin and IBMs (Fig. S2, Supplementary data).
16
The characteristic absorption regions around 3400 cm-1 can be assigned to the
17
vibrational band of the hydroxyl group of IBMs. However, a band shift from 3432
18
cm-1 to 3416 cm-1 is observed in the spectrum of CUR@IBMs powder compared with
19
that of IBMs, attributing to the formation of intermolecular hydrogen bonds between
20
the IBMs and curcumin[36].The result is in agreement with the formation mechanism
21
of co-assembly of C3Ms, which is mainly driven by hydrogen bonding and
22
hydrophobic interaction[26]. 16
Page 16 of 33
3.4. In vitro drug release
2
It has been reported that free curcumin would be quickly eliminated in the metabolic
3
process[37]. Nano-drug delivery systems may provide an alternative way to protect
4
curcumin from the metabolism before curcumin arrives the target sites. In this respect,
5
sustained release of curcumin is required. The release profiles of CUR@IBMs were
6
obtained in three media at pH 2.0, pH 5.0 and pH 6.8, simulating various
7
physiological environments. Three different pH values were selected in order to
8
simulate various physiological environments, including stomach (pH 1.0 ~ 2.5),
9
intestine (pH 6.4 ~ 7.5), blood and normal tissue (pH 7.4 ~ 7.5), intracellular
10
components (pH ranging from 4.5 in the lysosome to 8.0 in the mitochondria), and
11
solid tumor extracellular microenvironment (pH < 6.5)[38, 39]. As illustrated in Fig. 4,
12
curcumin was released much slower after being encapsulated by the IBMs compared
13
with free curcumin, which reveals sustained release of CUR@IBMs. For example,
14
after 72 h, 54%, 47% and 60% of the curcumin were released from the IBMs at pH
15
2.0, pH 5.0 and pH 6.8, respectively; by contrast, free curcumin had completed its
16
release at the three pHs. In addition, the CUR@IBMs themselves are highly
17
dispersible at the three pHs studied. The reason for the pH influence on the release of
18
IBMs may be due to the affinity of IBMs and curcumin. Previously, we have found
19
that polyphenols exhibit the strongest binding with the C3Ms at the isoelectric point
20
(pI) of gelatin (pI ≈ 5.0)[26], which could explain the least release of curcumin (47%)
21
from IBMs. While, the affinity would be weakened at pH 2.0 and pH 6.8, and led to
22
more curcumin release from the cores of the IBMs, about 54% and 60%, respectively.
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Page 17 of 33
It suggests that the CUR@IBMs can be used as sustained formulations applied either
2
in oral administration or in intravenous administration.
3
3.5. Cell cytotoxicity
4
The cell cytotoxicities of free curcumin, IBMs and CUR@IBMs were evaluated on
5
HeLa cells using MTT assay. The results revealed that both free curcumin and
6
CUR@IBMs display a concentration-dependent cytotoxicity against the HeLa cells,
7
while the IBMs show little cytotoxicity at the equivalent concentrations to the
8
CUR@IBMs. The encapsulated curcumin presents comparative or better cytotoxic
9
effects than free curcumin (see Fig. 5A).
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In addition, the IBMs show little cytotoxicity at the equivalent concentrations to the
11
CUR@IBMs towards normal L-929 cells (Fig. 5B). Meanwhile, CUR@IBMs showed
12
less cytotoxicity on the L-929 cells than on the HeLa cells. The result is in agreement
13
with other cell cytotoxicity studies of curcumin encapsulated by PLGA-MNPs and
14
N,O-carboxymethyl chitosan nanoparticles[40, 41], which may be due to the healthy
15
nature of the normal cells compared with the cancer cells.
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To further confirm the relationship between the anti-cancer activities and the cell
17
uptake, we observed the fluorescence of HeLa cells after incubation with free
18
curcumin and CUR@IBMs, respectively. The fluorescent images show that the
19
fluorescence of cells treated with free curcumin is relatively faint (Fig. 6). Whereas,
20
the fluorescence of HeLa cells is greatly enhanced, and it can be clearly observed in
21
the cytoplasm of HeLa cells after treated with CUR@IBMs. Our results are consistent
22
with other curcumin nanoparticulate formulations which were more effective than 18
Page 18 of 33
native curcumin against the growth of cancer cell lines due to the enhanced cell
2
uptake[42, 43]. In addition, L-929 cells incubated with CUR@IBMs showed faint
3
fluorescence intensity (Fig. 6), which implies that the less cell uptake of curcumin for
4
L-929 cells than that for HeLa cells, confirming the less cytotoxicity for L-929 cells.
5
3.6. In vivo oral bioavailability
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To understand the relative bioavailability of CUR@IBMs, plasma pharmacokinetic
7
after oral administration was studied on rat model by using free curcumin suspension
8
as control. The concentration of curcumin was monitored by fluorescence method.
9
Curcumin gives fluorescence emission at 520 nm when it is excited at 420 nm, which
10
has attracted many researches on fluorescent property of curcumin as a potential
11
fluorescent probe and photodynamic therapy drug[44, 45]. Fig. 7A shows the
12
fluorescence emission spectra of the detected curcumin in rat plasma samples after
13
oral administration for 1.5 h. Notably, the curcumin concentrations in rat plasma
14
exhibit significant increases following oral administration of 100 mg/kg CUR@IBMs
15
nanoparticles compared with 500 mg/kg free curcumin (Fig. 7A and 7B). In the
16
plasma of untreated rat, the fluorescence was negligible under the same excitation.
17
The results demonstrated the relative bioavailability of CUR@IBMs is higher than
18
that of free curcumin. Similar phenomena were observed in other curcumin
19
nanoformulation[14, 46]. As mentioned above, the bioavailability of curcumin is
20
limited by its low solubility, poor absorption, rapid degradation and excretion[3, 9].
21
The significant improvement of bioavailability in oral administration may be mainly
22
attributed to the higher absorption from the enhancement of solubility. Additionally,
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Page 19 of 33
1
the IBMs could protect curcumin against metabolism in the gastrointestinal tract and
2
in blood circulation before curcumin is released from nanoparticles, which could be
3
another reason attributed to the improvement of bioavailability.
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4. Conclusion
6
The novel IBMs were successfully prepared by a simple and green process and they
7
can specifically bind to polyphenol analogous drugs. High encapsulation of curcumin
8
in the IBMs facilitates complete dispersion of curcumin in water. The CUR@IBMs
9
possess the advantages of narrow size distribution, high drug encapsulation efficiency
10
and sustained release. Meanwhile, they display enhanced cell uptake, superior growth
11
inhibition against tumor cells and improved oral bioavailability over free curcumin,
12
indicating that the IBMs are potential nanovehicles for curcumin delivery in cancer
13
therapy.
14
Acknowledgements
15
This work was financially supported by the National Natural Science Foundation of
16
China (Nos. 21374133, 21276285 and 21104096), Hunan Provincial Natural Science
17
Foundation of China (No. 12JJ4014), China Postdoctoral Science Foundation (No.
18
2011M501281) and Postgraduate Science Foundation of Central South University
19
(No. 2013zzts161).
20
Appendix A. Supplementary data
21
Supplementary material related to this article can be found in the online version, at
22
http://dx.doi.org/10.1016/
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Fig. 2. (A) Binding capacity (Q) of the polyphenol analogous and non-analogous drugs in the IBMs and non-imprinted gelatin-dextran conjugates. (B) The specific binding (ΔQ) by the IBMs to polyphenol analogous and non-analogous drugs. Data of Q for each drug are statistically compared between IBMs and the conjugates using Student’s t-test in Microsoft Excel 2003. The level of significance is p < 0.05. Asterisk means a significant difference. Error bars for ΔQ represent the standard error (n = 3). Fig. 3. Size distributions of CUR@IBMs as-prepared and after lyophilization and then rehydration analyzed by DLS. Inset: Photos of free curcumin in water (left); curcumin and gelatin-dextran conjugates mixture (middle); CUR@IBMs (right). Fig. 4. Curcumin release profiles against different release medium buffers at pH 2.0 (A), pH 5.0 (B) and pH 6.8 (C). Fig. 5. Cell cytotoxicities of the IBMs, free curcumin and CUR@IBMs against HeLa cells (A) and 22
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L-929 cells (B) after 48 h incubation. The concentrations of IBMs are equivalent to those of CUR@IBMs. Asterisk means a significant difference (*p< 0.05).
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Scheme 1. Preparation of IBMs and CUR@IBMs.
Fig. 6. Bright and fluorescent microscopic images for the HeLa cell and L-929 cell uptake of free curcumin and CUR@IBMs after incubation for 2 h, respectively.
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Fig. 7. (A) The fluorescence emission spectra of CUR@IBMs in plasma (solid), free curcumin in plasma (dot), and untreated plasma (dash) after oral administration in rats for 1.5 h. Inset: The fluorescence emission spectra of free curcumin. (B) Concentration curve of curcumin in rat plasma after oral administration with free curcumin (500 mg/kg) and CUR@IBMs (100 mg/kg).
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Imprinted-like biopolymeric micelles (IBMs) for hydrophobic curcumin delivery were developed by using gelatin-dextran conjugate as an imprinting material and tea polyphenol (TPP) as a template.
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Highlights We prepared imprinted-like biopolymeric micelles (IBMs) via green process; IBMs showed imprinted-like property for the polyphenol analogous drugs; Water solubility of curcumin was increased by 5×104-fold after encapsulation; Curcumin-loaded IBMs exhibited superior growth inhibition against tumor cells; IBMs could effectively improve the oral bioavailability of curcumin.
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