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Formulation and in vitro Evaluation of Quercetin Loaded Polymeric Micelles
Composed of Pluronic P123 and TPGS
Liyan Zhao1,, Yikang Shi2,, Shaohua Zou3,, Min Sun1, Lingbing Li1, and Guangxi Zhai1*
1 Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan 250012, China
2 Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Science, Shandong
University, Jinan 250012, China
3
Department of Pharmacy, Yantai Yuhuangding Hospital, Yantai 26400, China
These authors contributed equally to the work
* Corresponding author:
Guangxi Zhai, Ph.D.
Professor
Department of Pharmaceutics
College of Pharmacy, Shandong University
44 Wenhua Xilu, Jinan 250012, China
Tel: (86) 531-8838,2015.
E-mail: [email protected]
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Abstract
The objective of this study was to develop a polymeric delivery system to improve the
solubility and biological activity of Quercetin (QT). QT loaded mixed micelles,
composed of Pluronic P123 (P123) and D-a-tocopheryl polyethylene glycol succinate
(TPGS) with proportion of 7:3 (QT-P/T), were prepared by thin-film hydration
method. The average size of the mixed micelles was 18.43 nm, and the encapsulating
efficiency for QT was 88.94 3.71%, drug-loading was 10.59 0.38%. The solubility
of QT in QT-P/T was 5.56 mg/mL, which was about 2738-fold that of crude QT in
water. Compared with the QT propylene glycol solution, the in vitro release of QT
from QT-P/T presented the sustained-release property. The in vitro cytotoxicity assay
showed that the IC50
values on MCF-7 cells for QT-P/T and QT loaded P123 micelles
(QT-P123) were 7.13 g/mL and 10.73 g/mL, respectively, while 7.23 g/mL and
14.47 g/mL on MCF-7/ADR cells. It could be concluded from the results that
P123/TPGS mixed micelles might serve as a pharmaceutical nanocarrier with
improved solubility and biological activity of QT.
Keywords: Quercetin, Polymeric Micelle, Pluronic P123, TPGS, Cytotoxicity
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INTRODUCTION
Quercetin (QT, Fig.1), extracted and isolated from Sophora japonica L, is the major
representative of the avonoid , and has a broad range of biological activities and
pharmacological actions such as anti-oxidant activity,1-2 anti-inammatory,3-4
anti-diabete,5 anti-neural disorders,6 anti-tumor and anti-proliferative effects on a
variety of human cancer cell lines.7-8 The in vitro and in vivo studies have
demonstrated that QT may inhibit cancer cell growth by binding to type II receptors,
which are over-expressed in a wide range of tumor tissues.9 In spite of this wide
spectrum of pharmacological properties, the clinical studies of QT have been
hampered due to the water insolubility (0.17-7.7 g/mL).10-12 In order to improve its
solubility, QT has been encapsulated in cyclodextrins,13 liposomes,14 and chitosan
nanoparticles.
Recent studies show that encapsulation of hydrophobic drugs inside polymeric
micelles is one of the most attractive alternatives.
15
16 Due to the nanosize and a
core-shell structure, polymeric micelles have been developed as drug delivery systems
for various agents in therapeutic and diagnostic applications,17-19 and as one promising
nanomedicine based technology, polymeric micelles as carriers for anticancer drugs
have been evaluated in several clinical trials.20-21 For example, SP1049C containing
doxorubicin in the mixed micelles of Pluronics L61 and F127 is the first anti-cancer
micellar formulation to reach clinic evaluation and is undergoing Phase II clinical
trials.
Pluronic P123 (P123), composed of PEO
22-23
20-PPO68-PEO20, is one of the most
common representatives of Pluronic copolymer.24 Here EO denotes oxyethylene
OCH2CH2, and PO denotes oxypropylene OCH2CH(CH3). It is a prominent feature
for P123 that can self-assemble into spherical micelle structure constructed by EO as
a hydrophilic outer shell and PO as a hydrophobic inner core.25 The PO core can serve
as a pool and the hydrophobic drug can be incorporated into the hydrophobic PO
core, while the hydrophilic corona maintains the dispersion stability of Pluronic
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micelles. It was also demonstrated that P123 had a significant cytotoxicity in the
multidrug resistant (MDR) cell lines to doxorubicin due to inhibition of the
P-glycoprotein (P-gp) drug efflux transport system that was over-expressed in these
cells.
D-a-tocopheryl polyethylene glycol succinate (TPGS) has been approved by FDA
as a water-soluble vitamin E nutritional supplement and drug delivery vehicle.
26
27 It is
reported that TPGS can enhance the solubility of poorly soluble drugs by micellar
solubilization.28 Recently, it is discovered that TPGS is one of P-gp inhibitory
excipients.29-30 Co-administrating with TPGS, the cellular uptake of doxorubicin on
Caco-2 cells is increased.31 And for amprenavir, a marketed antiviral drug, TPGS has
been used clinically to enhance the bioavailability of the drug.
In the present work, QT loaded P123/TPGS mixed micelles were prepared by
thin-film hydration method. The physicochemical properties and in vitro cytotoxicity
of the drug-loaded micelles were investigated.
32
MATERIALS AND METHODS
Materials
QT was purchased from Xian Senmu Biological Technology Co. Ltd (Xian, China).
TPGS was supplied by Wuhan Yuancheng Co. Ltd (Wuhan, China). P123,
3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), trypsin and
EDTA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human breast
carcinoma cell line MCF-7 and its adriamycin-resistant counterpart MCF-7/ADR
were donated by Institute of Biochemical and Biotechnological Drug, School of
Pharmaceutical Science, Shandong University. Penicillin streptomycin, RPMI 1640
and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersberg, MD,
USA). All other chemicals were of analytical grade.
Preparation of QT Loaded Micelles
QT loaded micelles were prepared by thin-film hydration method.33-34 Briefly, 24mg
of QT and 10 mM of copolymer carriers composed of P123 and TPGS with different
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proportions were dissolved in alcohol. The solution was subsequently evaporated
under reduced pressure by rotary vacuum evaporation to obtain a thin film of
drug/polymer mixture, and the film was further dried over night at room temperature
to remove any residual. After that, the obtained film was hydrated in 4mL of
de-ionized water under magnetic stirring at room temperature to form a micellar
suspension. Non-incorporated crystalline drug was separated by filtration through a
0.22 m lter membrane , and a yellow clear solution of QT loaded mixed micelles
was obtained.
QT loaded P123 micelles (QT-P123) and empty micelles were prepared according
to the same procedure.
Characterization of Micelles
Particle Size Distribution
Particle size distributions and mean diameters of the prepared micelles were measured
using the BI-200SM based on the light dynamic scattering method (DLS, Brookhaven
Instruments Corporation, USA) at a scattering angle of 90 at room temperature. Each
freshly prepared sample was placed into a quartz cuvette without additional treatment.
The size distributions were extracted from the autocorrelation functions by the
CONTIN program.36
For each sample, the size was measured in triplicate.
Surface Morphology
The morphology of the QT-P/T was observed under transmission electron microscope
(TEM, JEM-1200EX, JEOL, Tokyo, Japan), and the accelerating voltage was 100 kv.
To prepare the TEM samples, a drop of micellar solution was placed on a copper grid
and stained with phosphotungstic acid solution (2%, w/v) about 15 s. Subsequently
the sample was allowed to dry slowly in air and then examined under TEM.
Zeta-potential
Zeta-potential of different micelle solution was determined using Zeta Potential
Analyzer Instrument (Brookhaven Instruments Corporation, USA). For each sample,
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zeta-potential measurement was repeated eight times.
Drug-loading and Entrapment Efficiency
The concentration of QT in the micelles was determined with UV-Vis
spectrophotometer (UV-2102, Shanghai Instrument Ltd, China) at a wavelength of
374 nm. The micellar solution was suitably diluted with alcohol prior to determination.
The drug-loading (DL%) and entrapment efficiency (EE%) of QT in polymeric
micelles were calculated from the following equations:
DL% =
24,37
weight of the drug in micellesweight of the feeding polymer and drug
EE% =
100% (1)
weight of the drug in micellesweight of the feeding drug
100% (2)
Critical Micelle Concentration (CMC)
In this study, CMC was analyzed by a fluorescence probe technique using pyrene as a
hydrophobic probe.38-40 The concentration of encapsulated pyrene in micelle phase
was determined using F-2500 fluorescence spectrometer (Hitachi, Japan). Pyrene
dissolved in acetone was added to empty vials. After acetone evaporation, a series of
micellar solutions were added to the vials. The final pyrene concentration was 6.0
107
M, slightly below the saturation concentration of pyrene in water at 25 C, and
the mixed solution was incubated overnight in the dark. All samples were excited at
334 nm, and fluorescence spectra were recorded between 350 nm and 500 nm. The
excitation and emission slit widths were set at 5 nm. Upon formation of micelles,
pyrene would move into the inside of the micelles from the aqueous phase, which
could result in an alteration in the intensity ratio of I372/I383.
Solubility of QT in Water or Polymer Micelle Solution
The solubility of QT in water was determined as follows, excessive crude QT powder
was added to 10 mL of de-ionized water, and then the resulting mixture was stirred at
100 rpm at 25 C for 72 h and centrifuged at 10000 rpm for 15 min. The supernatant
was taken and filtrated through a 0.22 m lter membrane, and subsequent ly, the
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content of QT in the obtained filtrate was analyzed with UV method at 374 nm.
1 mL of QT loaded micelle solution was suitably diluted with alcohol, and then the
resulting solution was analyzed with UV method at 374 nm, the concentration of QT
in micelle solution was calculated and the solubility of QT in polymer micelle
solution was obtained.
In vitro Release of QT from Micelles
The release of QT from the micelles was investigated by dialysis method with 0.5%
Tween 80 solution as release medium. The solution containing 3.0 mg of QT was
introduced into a pre-swollen dialysis tube with a MWCO of 8KD-12KD (Xian
Luosenbo Co. Ltd, Xian, China), and the dialysis tube was immersed into 200 mL
release medium at 370.5C with stirring speed at 100 rpm. At predetermined time
intervals, 4 mL of the dissolution medium was withdrawn and the same volume of
fresh medium was added. Then, the amount of released QT was measured by UV-Vis
spectrophotometer at 374 nm, and the cumulative release percentage (Q%) was
calculated. For comparison, the release of QT from propylene glycol solution was
conducted under the same conditions.
41
Cytotoxicity in vitro
The cytotoxicity in vitro of drug loaded micelles was evaluated on MCF-7 and
MCF-7/ADR using the MTT method.30,34,42 The cells were cultured in RPMI-1640
medium, which was supplemented with 2 mM l-glutamine, 10% (v/v) FBS, 100
units/mL penicillin G, 0.25 g/mL amphotericin B, and 100 g/mL streptomicin at 37
C in a humidified 5% CO2
MCF-7 cell lines were seeded at the density of 310
sterile incubator. The medium was changed once every
two days. 3 cells per well in 96-well
plates and 8103 cells per well for MCF-7/ADR cells. After 24 h incubation, 100 L
of medium containing the treatment agents such as QT DMSO solution (the final
concentration of DMSO kept below 0.2%), empty micellar solutions and drug loaded
micellar solutions of various concentrations was added. The concentration of QT
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ranged from 0.5 to 60 g/mL. After additional 48 h incubation, the cells were washed
twice with phosphate buffer saline (PBS). Subsequently, the growth medium was
refreshed and 20 L of MTT solution (5 mg/mL) was added to each well. The plates
were incubated at 37 C for another 4 h and the medium was removed again. The
intracellular metabolized product formazan crystals were dissolved by addition of 150
L of DMSO to each well. The absorbance was measured using a multiwell scanning
spectrophotometer Model 680 (Bio-Rad, USA) with the test wavelength at 570 nm
and the reference wavelength at 630 nm. Cell viability was calculated by [absorbance
of cells exposed to micelles or drug] / [absorbance of cells cultured without micelles
or drug] in percentage.
RESULTS AND DISCUSSION
Particle Size Distribution
The particle size will directly affect the bio-distribution and circulation time in vivo of
the carriers.43
The micelle size obtained by DLS depends on both the block copolymer
composition and the drug loading. After the micelle was formed, the micelle size was
mainly influenced by the interaction of the hydrophobic fractions. The mean diameter
of QT-P/T (18.43 nm) was smaller than that of QT-P123 (29.04 nm). This might be
attributed to the influence of copolymer composition, in copolymer carrier kept
constant at 10 mM, part of P123 molecules was replaced by TPGS with a smaller
molecular weight and a smaller hydrophobic group than P123, so QT-P/T showed a
significantly smaller hydrophobic volume than that of QT-P123, which might result in
The average size and size distribution for empty micelles and drug
loaded micelles were presented in Fig.2. The average size of empty P123 micelles and
mixed micelles composed of P123/TPGS was under 10 nm with rather narrow size
distribution patterns. An increasing in the average size after QT loading was observed
from 8.85 nm (Fig.2(C)) to 29.04 nm (Fig.2(A)) for P123 micelles and from 9.45 nm
(Fig.2(D)) to 18.43 nm (Fig.2(B)) for mixed micelles composed of P123/TPGS with
the molar ratio at 7:3, respectively.
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smaller size.
Stable and small particle sizes (
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block length, the nature and block length of the outer shell for micelle.46
The solubility of QT in the obtained QT-P/T micelle solution was 5.56 0.34
mg/mL, while only 2.03 0.44 g/mL for that of QT in water. That is to say, the
solubility of QT in the polymeric micelles was about 2738-fold that of crude QT in
water.
Based on
these reasons, copolymer carriers composed of P123 and TPGS with different
proportions were studied. As shown in Table 1, the DL% of the QT-P/T with the molar
ratio of P123 to TPGS at 7:3 (10.59 0.38%) was higher than that of QT-P123 (8.25
0.32%). This result might be related to the stable reaction among the aromatic ring
in TPGS, PO groups in P123 and incorporated drug. However, when the proportion
was at 5:5, the EE% (37.95 3.27%) and DL% (5.84 0.50%) were markedly
decreased. The possible reason was that QT-P/T with the molar ratio of P123 to TPGS
at 5:5 showed a significantly smaller hydrophobic volume than that of QT-P123 or
QT-P/T with the molar ratio of P123 to TPGS at 7:3.
Critical Micelle Concentration (CMC)
Pluronic copolymers consist of ethylene oxide (EO) and propylene oxide (PO) blocks,
and can undergo self-assembly into spherical micelles in aqueous solution.40
The CMC value for micellar solution made from P/T with the molar ratio of P123
to TPGS at 7:3 or P123 was as low as 1.9310
CMC
was a parameter indicative of the micelles stability in vitro and in vivo. In this study,
it was measured by fluorescence technique with the pyrene as a hydrophobic probe.
The CMC value was obtained by plotting the ratio of I372/I383 of the emission
spectra profile vs the concentration of copolymers as shown in Fig.6. This ratio of
I372/I383 was decreased with increasing the concentration of copolymer.
-5 M (Fig.6 (A)) or 1.9710-5 M (Fig.6
(B)), respectively, which was in accordance with previous report.47
The addition of
TPGS did not result in notable variation in the CMC. Because of the low CMC, the
micelles had high stability and ability to maintain integrity even upon extreme
dilution in body.
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In vitro Release of QT from Micelles
The in vitro release of QT from micellar formulation under sink condition was
investigated by dialysis method with 0.5% Tween 80 solution as release medium. As
shown in Fig.7, only 15% of QT was released from QT-P/T and QT-P123 within the
first 4 h, while almost all QT was released from the propylene glycol solution during
the same time period. After 60 h, 20-30% of the initially incorporated drug still
existed in the micelles. The result indicated that the micelles showed a
sustained-release property for the incorporated QT, which was similar to the reported
studies.33,48,49 The released mechanism of QT from micelles might be related to the
drug diffusion and the polymer material erosions or swelling.50 It was noticed that the
release of QT from mixed micelles was faster than that of P123 micelles. It could be
explained that the addition of TPGS enlarged the ratio of hydrophilic part in the
mixed micelles and facilitated water molecules into the core of the micelles, leading
to more hydrophilic channels.
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Cytotoxicity in vitro
The in vitro cytotoxicity of QT-P/T and QT-P123 was assessed on MCF-7 and
MCF-7/ADR cells with QT DMSO solution as control. The empty micelles of P/T
and P123 with the same copolymer concentrations as QT-P/T and QT-P123 were used
as control, too. The cells were incubated for 48 h in the presence of the micelles or
free QT, and then their survival was analyzed using the MTT assay. The viability of
MCF-7 and MCF-7/ADR cells after incubation with various formulations of QT and
empty micelles was presented in Fig.8. The IC50
It was reported that P123 did not display obvious cytotoxicity to HDF broblast
cells.
values on MCF-7 cells for free QT in
DMSO solution, QT-P/T, and QT-P123 were 16.32 g/mL, 7.13 g/mL and 10.73
g/mL, respectively, while 16.87 g/mL, 7.23 g/mL and 14.47 g/mL on
MCF-7/ADR cells (Fig.8 (A,C)). The results demonstrated that QT-P/T showed a
higher cytotoxicity compared to the free drug and QT-P123 on both MCF-7 and
MCF-7/ADR cells.
51 However, in this study, it was obvious that the empty micelles of P/T and P123
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displayed cytotoxicity on MCF-7 and MCF-7/ADR cell lines, and the cytotoxicity of
P/T empty micelles was higher than that of P123 empty micelles (Fig.8 (B,D)). This
could be explained with addition of the TPGS. TPGS in the mixed micelles might act
as P-glycoprotein inhibitor to reduce drug efflux. Moreover, animal studies of human
cancer xenografts found that TPGS could effectively suppress tumor growth. The
anticancer activity of TPGS was reported to be related to its unique apoptosis-
inducing properties via the generation of reactive oxygen species (ROS). ROS could
damage DNA, proteins, and fatty acids in cells, resulting in apoptotic cell death.30
Therefore, QT-P/T showed higher cytotoxicity and might be considered as an
effective anticancer drug delivery system for cancer chemotherapy compared with
QT-P123.
CONCLUSIONS
The mixed polymeric micelles, composed of P123 and TPGS with the proportion of
7:3, exhibited higher encapsulating efficiency and drug-loading for QT. The average
size of QT loaded mixed micelles was 18.43 nm, and zeta potential was -10.18 mV.
The solubility of QT in QT-P/T was 5.56 mg/mL, which was about 2738-fold that of
crude QT in water. Compared with the free drug, QT-P/T showed a significantly
enhanced cytotoxicity. Based on these results, it can be concluded that the polymeric
micelles formulation developed in this study may be considered as a promising
delivery system for QT.
Acknowledgements: This work is partly supported by a research grant
(No.2008GG10002012) from Department of Shandong Science and technology, P. R.
China.
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REFERENCES
1. K. Ioku, T. Tsushida, Y. Takei, N. Nakatani, and J. Terao, Antioxidative activity of
quercetin and quercetin monoglucosides in solution and phospholipid bilayers.
Biochim. Biophys. Acta 1234, 99 (1995).
2. H. Maria, S. M. Concepcin, and P. T. Sonia, Flavonoid-avonoid interaction and
its effect on their antioxidant activity. Food Chem. 121, 691 (2010).
3. A. Gomes, E. Fernandes, J. L. Lima, L. Mira, and M. L. Corvo, Molecular
mechanisms of anti-inflammatory activity mediated by flavonoids. Curr. Med.
Chem. 15, 1586 (2008).
4. A. P. Rogerio, C. L. Dora, E. L. Andrade, J. S. Chaves, L. F. Silva, E.
Lemos-Senna, and J. B. Calixto, Anti-inflammatory effect of quercetin-loaded
microemulsion in the airways allergic inflammatory model in mice. Pharmacol.
Res. 61, 288 (2010).
5. X. K. Fang, J. Gao, and D.N. Zhu, Kaempferol and quercetin isolated from
Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis
activity. Life Sci. 82, 615 (2008).
6. B. Ossola, T. M. Kaariainen, and P. T. Mannisto. The multiple faces of quercetin
in neuroprotection. Expert Opin. Drug Saf. 8, 397 (2009).
7. K. V. Hirpara, P. Aggarwal, A. J. Mukherjee, N. Joshi, and A. C. Burman,
Quercetin and its derivatives: synthesis, pharmacological uses with special
emphasis on anti-tumor properties and prodrug with enhanced bio-availability.
Anticancer Agents Med. Chem. 9, 138 (2009).
8. G. Scambia, F. O. Ranelletti, P. Benedetti Panici, M. Piantelli, G. Bonanno, R. De
Vincenzo, G. Ferrandina, L. Pierelli, A. Capelli, and S. Mancuso, Quercetin
inhibits the growth of a multidrug-resistant estrogen-receptor-negative MCF-7
human breast-cancer cell line expressing type II estrogen-binding sites. Cancer
Chemother. Pharmacol. 28, 255 (1991).
9. M. E. Ribeiro, I. G. Vieira, I. M. Cavalcante, N. M. Ricardo, D. Attwood, S. G.
Yeates, and C. Booth, Solubilisation of griseofulvin, quercetin and rutin in
-
14
micellar formulations of triblock copolymers E62P39E62 and E137S18E137. Int.
J. Pharm. 378, 211 (2009).
10. Y. Gao, Y. Wang, Y. Ma, A. Yu, F. Cai, W. Shao, and G. Zhai, Formulation
optimization and in situ absorption in rat intestinal tract of quercetin-loaded
microemulsion. Colloids Surf. B 71, 306 (2009).
11. M. Sun, Y. Gao, Y. Pei, C. Y. Guo, H. Li, F. Cao, A. Yu, and G. X. Zhai,
Development of nanosuspension formulation for oral delivery of quercetin. J.
Biomed. Nanotechnol. 6, 325 (2010).
12. N. Bernardy, A. P. Romio, E. I. Barcelos, C. Dal Pizzol, C. L. Dora, E.
Lemos-Senna, P. H. Araujo, and C. Sayer, Nanoencapsulation of quercetin via
miniemulsion polymerization. J. Biomed. Nanotechnol. 6, 181 (2010).
13. T. Pralhad and K. Rajendrakumar, Study of freeze-dried quercetin-cyclodextrin
binary systems by DSC, FT-IR, X-ray diffraction and SEM analysis. J. Pharm.
Biomed. Anal. 34, 333 (2004).
14. A. Priprem, J. Watanatorn, S. Sutthiparinyanont, W. Phachonpai, and S.
Muchimapura, Anxiety and cognitive effects of quercetin liposomes in rats.
Nanomedicine 4, 70 (2008).
15. Y. Zhang, Y. Yang, K. Tang, X. Hu, and G. Zou, Physicochemical
characterization and antioxidant activity of quercetin-loaded chitosan
nanoparticles. J. Appl. Polym. Sci. 107, 891 (2008).
16. D. Chiappetta and A. Sosnik, Poly(ethylene oxide)-poly(propylene oxide) block
copolymer micelles as drug delivery agents: Improved hydrosolubility, stability
and bioavailability of drugs. Eur. J. Pharm. Biopharm. 66, 303 (2007).
17. L. E. Vlerken and M. M. Amiji, Multi-functional polymeric nanoparticles for
tumour-targeted drug delivery. Expert Opin. Drug Deliv. 3, 205 (2006).
18. N. Nasongkla, E. Bey, J. Ren, H. Ai, C. Khemtong, J. S. Guthi, S. F. Chin, A. D.
Sherry, D. A. Boothman, and J. Gao, Multifunctional polymeric micelles as
cancer-targeted, MRI-ultrasensitive drug delivery systems. Nano. Lett. 6, 2427
(2006).
19. C. Sun, R. Sze, and M. Zhang, Folic acid-PEG conjugated supermagnetic
-
15
nanoparticles for targeted cellular uptake and detection. J. Biomed. Mater. Res.
78A, 550 (2006).
20. Y. Mizumur, Y. Matsumur, M. Yokoyam, T. Okano, T. Kawaguchi, F. Moriyasu,
and T. Kakizoe, Incorporation of the anticancer agent KRN5500 into polymeric
micelles diminishes the pulmonary toxicity. Jpn. J. Cancer Res. 93, 1237 (2002).
21. Y. Matsumura, T. Hamaguchi, T. Ura, K. Muro, Y. Yamada, Y. Shimada, K.
Shirao, T. Okusaka, H. Ueno, M. Ikeda, and N. Watanabe, Phase I clinical trial
and pharmacokinetic evaluation of NK911, a micelle-encapsulated doxorubicin.
Br. J. Cancer 91, 1775 (2004).
22. S. Danson, D. Ferry, V. Alakhov, J. Margison, D. Kerr, D. Jowle, M. Brampton,
G. Halbert, and M. Ranson, Phase I dose escalation and pharmacokinetic study of
pluronic polymer-bound doxorubicin (SP1049C) in patients with advanced
cancer. Br. J. Cancer 90, 2085 (2004).
23. E. V. Batrakova, A. V. Kabanov, Pluronic block copolymers: evolution of drug
delivery concept from inert nanocarriers to biological response modifiers. J.
Control. Release 130, 98 (2008).
24. J. Jansson, K. Schille, G. Olofsson, R. Cardoso, and W. Loh, The Interaction
between PEO-PPO-PEO Triblock Copolymers and Ionic Surfactants in Aqueous
Solution Studied Using Light Scattering and Calorimetry. J. Phys. Chem. B 108,
82 (2004).
25. T. Nakanishi, S. Fukushima, K. Okamoto, M. Suzuki, Y. Matsumura, M.
Yokoyama, T. Okano, Y. Sakurai, and K. Kataoka, Development of the polymer
micelle carrier system for doxorubicin. J. Control. Release 74, 295 (2001).
26. A. V. Kabanov, E. V. Batrakova, and D. W. Miller, Pluronic block copolymers as
modulators of drug efflux transporter activity in the blood-brain barrier. Adv.
Drug Deliv. Rev. 55, 151 (2003).
27. A. Yan, A. Von Dem Bussche, A. B. Kane, and R. H. Hurt, Tocopheryl
Polyethylene Glycol Succinate as a Safe, Antioxidant Surfactant for Processing
Carbon Nanotubes and Fullerenes. Carbon. N. Y. 45, 2463 (2007).
28. M. T. Sheu, S. Y. Chen, L. C. Chen, and H. O. Ho, Influence of micelle
-
16
solubilization by tocopheryl polyethylene glycol succinate (TPGS) on solubility
enhancement and percutaneous penetration of estradiol. J. Control. Release 88,
355 (2003).
29. T. Bansal, N. Akhtar, M. Jaggi, R. K. Khar, and S. Talegaonkar, Novel
formulation approaches for optimising delivery of anticancer drugs based on
P-glycoprotein modulation. Drug Discov. Today 14, 1067 (2009).
30. H. Zhao, L. Y. Yung, Addition of TPGS to folate-conjugated polymer micelles
for selective tumor targeting. J. Biomed. Mater. Res. A. 91, 505 (2009).
31. E. M. Collnot, C. Baldes, M. F. Wempe, J. Hyatt, L. Navarro, K. J. Edgar, U. F.
Schaefer, and C. M. Lehr, Influence of vitamin E TPGS poly(ethylene glycol)
chain length on apical efflux transporters in Caco-2 cell monolayers. J. Control.
Release 111, 35 (2006).
32. L. Yu, A. Bridgers, J. Polli, A. Vickers, S. Long, A. Roy, R. Winnike, and M.
Coffin, Vitamin E-TPGS increases absorption flux of an HIV protease inhibitor
by enhancing its solubility and permeability. Pharm. Res. 16, 1812 (1999).
33. Y. Gao, L. B. Li, and G. X. Zhai, Preparation and characterization of
Pluronic/TPGS mixed micelles for solubilization of camptothecin. Colloids Surf.
B 64, 194 (2008).
34. R. D. Dabholkar, R. M. Sawant, D. A. Mongayt, P. V. Devarajan, and V. P.
Torchilin, Polyethylene glycol-phosphatidylethanolamine conjugate
(PEG-PE)-based mixed micelles: some properties, loading with paclitaxel, and
modulation of P-glycoprotein-mediated efflux. Int. J. Pharm. 315, 148 (2006).
35. Z. Wei, J. Hao, S. Yuan, Y. Li, W. Juan, X. Sha, and X. Fang, Paclitaxel-loaded
Pluronic P123/F127 mixed polymeric micelles: formulation, optimization and in
vitro characterization. Int. J. Pharm. 376, 176 (2009).
36. R. Ganguly, V. K. Aswal, and P. A. Hassan, Room temperature sphere-to-rod
growth and gelation of PEO-PPO-PEO triblock copolymers in aqueous salt
solutions. J. Colloid Interface Sci. 315, 693 (2007).
37. J. Fu, D. Wang, T. Wang, W. Yang, Y. Deng, H. Wang, S. Jin, and N He, High
entrapment efficiency of chitosan/polylactic acid/tripolyphotspate nanosized
-
17
microcapsules for rapamycin by an emulsion-evaporation approach. J. Biomed.
Nanotechnol. 6, 725 (2010).
38. K. H. Bae, S. H. Choi, S. Y. Park, Y. Lee, and T. G. Park, Thermosensitive
pluronic micelles stabilized by shell cross-linking with gold nanoparticles.
Langmuir 22, 6380 (2006).
39. J. Gong, M. Huo, J. Zhou, Y. Zhang, X. Peng, D. Yu, H. Zhang, and J. Li,
Synthesis, characterization, drug-loading capacity and safety of novel octyl
modified serum albumin micelles. Int. J. Pharm. 376, 161 (2009).
40. X. Yang, L. Li, Y. Wang, and Y. Tan, Preparation, pharmacokinetics and tissue
distribution of micelles made of reverse thermo-responsive polymers. Int. J.
Pharm. 370, 210 (2009).
41. H. Li, X. Zhao, Y. Ma, G. Zhai, L. Li, and H. Lou, Enhancement of
gastrointestinal absorption of quercetin by solid lipid nanoparticles. J. Control.
Release 133, 238 (2009).
42. P. Wu, X. He, K. Wang, W. Tan, C. He, and M. Zheng, A novel methotrexate
delivery system based on chitosan-methotrexate covalently conjugated
nanoparticles. J. Biomed. Nanotechnol. 5, 557 (2009).
43. M. Prabaharan, J. J. Grailer, S. Pilla, D. A. Steeber, and S. Gong, Amphiphilic
multi-arm-block copolymer conjugated with doxorubicin via pH-sensitive
hydrazone bond for tumor-targeted drug delivery. Biomaterials 30, 5757 (2009).
44. T. Wang, N. He, Preparation, characterization and applications of
low-molecular-weight alginate-oligochitosan nanocapsules. Nanoscale 2, 230
(2010).
45. W. Liu, R. Guo, Interaction between flavonoid, quercetin and surfactant
aggregates with different charges. J. Colloid Interface Sci. 302, 625 (2006).
46. Z. Sezgin, N. Yuksel, and T. Baykara, Preparation and characterization of
polymeric micelles for solubilization of poorly soluble anticancer drugs. Eur. J.
Pharm. Biopharm. 64, 261 (2006).
47. D. Lof, M. Tomsic, O. Glatter, G. Fritz-Popovski, and K. Schilln, Structural
characterization of nonionic mixed micelles formed by C12EO6 surfactant and
-
18
P123 triblock copolymer. J. Phys. Chem. B. 113, 5478 (2009).
48. J. Wang, R. Wang, and L. B. Li, Preparation and properties of
hydroxycamptothecin-loaded nanoparticles made of amphiphilic copolymer and
normal polymer. J. Colloid Interface Sci. 336, 808 (2009).
49. W. Yang, J. Fu, T. Wang, and N. He, Chitosan/sodium tripolyphosphate
nanoparticles: preparation, characterization and application as drug carrier. J.
Biomed. Nanotechnol. 5, 591 (2009).
50. K. S. Kim, S. J. Park, Effect of porous silica on sustained release behaviors of
pH sensitive Pluronic F127/poly(acrylic acid) hydrogels containing tulobutero.
Colloids Surf. B 80, 240 (2010).
51. H. Song, R. He, K. Wang, J. Ruan, C. Bao, N. Li, J. Ji, and D. Cui,
Anti-HIF-1alpha antibody-conjugated pluronic triblock copolymers encapsulated
with Paclitaxel for tumor targeting therapy. Biomaterials 31, 2302 (2010).
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Table 1 EE% and DL% of QT in micelles at 10 mM copolymers (n = 5)
Composition (molar ratio) EE% DL%
P123 (10) 87.06 3.80 8.25 0.32
P123:TPGS (5:5) 37.95 3.27 5.84 0.50
P123:TPGS (7:3) 88.94 3.71 10.59 0.38
Liyan Zhao, et al., Table 1
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20
Captions
Fig.1 The structure of QT.
Fig.2 DLS particle size distribution of QT-P123 (A), QT-P/T (B), empty P123
micelles (C), and empty P/T mixed micelles (D).
Fig.3 TEM image of QT loaded P123/TPGS mixed micelles (19,0000).
Fig.4 Schematic illustration of QT loaded micelle composed of P123 and TPGS.
Fig.5 Photographic images of QT-P/T (A), QT-P123 (B) and QT suspension (C).
Fig.6 Plot of I372/I383 vs concentrations of copolymers in deionized water.
Copolymers of P123/TPGS (7:3) (A); P123 (B)
Fig.7 Release proles of QT from QT-P123 (), QT-P/T () and the propylene glycol
solution () in 0.5% Tween 80 solution at 37 C. Each point represents average
SD (n = 3).
Fig.8 Viability of MCF-7 cells after incubation with various formulations of QT (A),
and empty micelles (B); Viability of MCF-7/ADR cells after incubation with
various formulations of QT (C), empty micelles (D). Each point represents
average SD (n = 3).
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21
Fig.1 The structure of QT.
Liyan Zhao, et al., Fig. 1
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22
20 25 30 35 40 450.0
0.1
0.2
0.3
0.4
0.5
A
in cl
ass
Diameter (nm)10 15 20 25 30 35
0.00
0.05
0.10
0.15
0.20
0.25 B
in cl
ass
Diameter (nm)
10 15 20 25 30 350.00
0.05
0.10
0.15
0.20
0.25 B
in cl
ass
Diameter (nm)
4 6 8 10 12 14 16 18 200.00
0.05
0.10
0.15
0.20
0.25 D
in cl
ass
Dimameter (nm)
Fig.2 DLS particle size distribution of QT-P123 (A), QT-P/T (B), empty P123
micelles (C), and empty P/T mixed micelles (D).
Liyan Zhao, et al., Fig. 2
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23
Fig.3 TEM image of QT loaded P123/TPGS mixed micelles (19,0000).
Liyan Zhao, et al., Fig. 3
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24
Fig.4 Schematic illustration of QT loaded micelle composed of P123 and TPGS.
Liyan Zhao, et al., Fig. 4
-
25
Fig.5 Photographic images of QT-P/T (A), QT-P123 (B) and QT suspension (C).
Liyan Zhao, et al., Fig. 5
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26
11.11.21.31.41.51.61.71.8
0 20 40 60 80 100 120 140 160
mM/mL
I372
/I383
A
11.11.21.31.41.51.61.71.8
0 20 40 60 80 100 120 140 160
mM/mL
I372
/I383
11.11.21.31.41.51.61.71.8
0 20 40 60 80 100 120 140 160
mM/mL
I372
/I383
A
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
0 20 40 60 80 100 120 140 160
mM/mL
I372/I383
B
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
0 20 40 60 80 100 120 140 160
mM/mL
I372/I383
B
Fig.6 Plot of I372/I383 vs concentrations of copolymers in deionized water.
Copolymers of P123/TPGS (7:3) (A); P123 (B)
Liyan Zhao, et al., Fig. 6
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27
0
20
40
60
80
100
120
0 10 20 30 40 50 60
Time (h)
Cum
ulat
ive
rele
ase
(% free QTQT-P/TQT-P123
Fig.7 Release proles of QT from QT-P123(), QT-P/T() and the propylene glycol
solution () in 0.5% Tween 80 solution at 37 C. Each point represents average
SD (n = 3).
Liyan Zhao, et al., Fig. 7
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28
Fig.8 Viability of MCF-7 cells after incubation with various formulations of QT (A), and empty micelles (B); Viability of MCF-7/ADR cells after incubation with various
formulations of QT (C), empty micelles (D). Each point represents average SD (n =
3).
Liyan Zhao, et al., Fig. 8
A
0
20
40
60
80
100
120
0 20 40 60 80
Concentration (g/ml)
Cel
l via
bilit
y QT-P/TQT-P123 free QT
B
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70 80
Concentration (g/ml)
Cel
l via
bilit
y
empty P/T empty P123
C
0
20
40
60
80
100
120
0 20 40 60 80
Concentration (g/mL)
Cel
l via
bilit
y
QT-P/T QT-P123free QT
D
0
20
40
60
80
100
120
0 20 40 60 80
Concentration (g/ml)
Cel
l via
bilit
y
empty QT-P/T
empty QT-P123
Materials and MethodsMaterialsPreparation of QT Loaded MicellesCharacterization of MicellesParticle Size DistributionDrug-loading and Entrapment EfficiencyCritical Micelle Concentration (CMC)In vitro Release of QT from MicellesCytotoxicity in vitroParticle Size DistributionSurface Morphology and Zeta-potentialBoth QT-P123 and QT-P/T were negatively charged with zeta-potential of about -4.08 mV and -10.18 mV, respectively. While the empty P123 micelles were almost neutral (0.78 mV) and the zeta-potential of the empty P/T micelles was -7.48 mV. The structu...Critical Micelle Concentration (CMC)In vitro Release of QT from MicellesCytotoxicity in vitroConclusionsReferences