1

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: professorgxzhai@yahoo.cn

2

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 μg/mL and 10.73 μg/mL, respectively, while 7.23 μg/mL and

14.47 μg/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

3

INTRODUCTION

Quercetin (QT, Fig.1), extracted and isolated from Sophora japonica L, is the major

representative of the flavonoid , and has a broad range of biological activities and

pharmacological actions such as anti-oxidant activity,1-2 anti-inflammatory,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 μg/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

4

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 Xi’an Senmu Biological Technology Co. Ltd (Xi’an, 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

5

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 μm filter 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,

6

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×

10−7

M, slightly below the saturation concentration of pyrene in water at 25 ºC, 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 ºC for 72 h and centrifuged at 10000 rpm for 15 min. The supernatant

was taken and filtrated through a 0.22 μm filter membrane, and subsequent ly, the

7

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 (Xi’an

Luosenbo Co. Ltd, Xi’an, China), and the dialysis tube was immersed into 200 mL

release medium at 37±0.5ºC 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 μg/mL amphotericin B, and 100 μg/mL streptomicin at 37

ºC in a humidified 5% CO2

MCF-7 cell lines were seeded at the density of 3×10

sterile incubator. The medium was changed once every

two days. 3 cells per well in 96-well

plates and 8×103 cells per well for MCF-7/ADR cells. After 24 h incubation, 100 μL

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

8

ranged from 0.5 to 60 μg/mL. After additional 48 h incubation, the cells were washed

twice with phosphate buffer saline (PBS). Subsequently, the growth medium was

refreshed and 20 μL of MTT solution (5 mg/mL) was added to each well. The plates

were incubated at 37 ºC for another 4 h and the medium was removed again. The

intracellular metabolized product formazan crystals were dissolved by addition of 150

μL 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.

9

smaller size.

Stable and small particle sizes (<200 nm) could reduce the uptake of the

reticuloendothelial system (RES) and provide efficient passive tumor-targeting ability

via the enhanced permeability and retention effects.

44

33

Therefore, the size of prepared

micelles was suitable for tumor-specific accumulation via the EPR effect.

Surface Morphology and Zeta-potential

As seen from Fig.3, the mixed micelles presented nanometric and homogeneous

spheres, and a core-shell structure was also observed under TEM. The outer shell

might be attributed to the hydrophilic PEG in TPGS and PEO in P123, and inner core

should relate to hydrophobic PPO and Vitamin E. The possible organization of the

mixed micelles made from copolymer P123 and TPGS in water through the

hydrophobic interaction was depicted schematically in Fig.4. And the obtained QT

loaded micellar solutions were clear and yellowish as shown in Fig.5 with the

suspension of crude QT dispersed in water at the same drug concentration as control.

Both 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

structure of P123, both the PPO and PEO segments, was nonionic, so the change of

surface charge of the micelles should result from addition of drug and TPGS. As we

know, the most acidic phenolic OH groups of QT can dissociate and result in the

anionic property for QT.45

Therefore, the absolute value of zeta-potential was

increased as addition of drug. Compared with QT-P123, an increase in the absolute

value of zeta-potential for QT-P/T was observed, too. The increase of negative surface

charge of QT-P/T might be attributed to TPGS which enhanced the solubility of QT

and more anionic QT presented in solution.

Incorporation of QT in Micelles

The major factors which influenced the DL% and EE% of copolymer micelles were

the nature and concentration of the solute, nature of the core forming block, core

10

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 μg/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 1.93×10

CMC

was a parameter indicative of the micelle’s 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 1.97×10-5 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.

11

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.

35

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 fibroblast

cells.

values on MCF-7 cells for free QT in

DMSO solution, QT-P/T, and QT-P123 were 16.32 μg/mL, 7.13 μg/mL and 10.73

μg/mL, respectively, while 16.87 μg/mL, 7.23 μg/mL and 14.47 μg/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

12

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.

13

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19

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

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 profiles of QT from QT-P123 (▲), QT-P/T (■) and the propylene glycol

solution (◆) in 0.5% Tween 80 solution at 37 ºC. 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).

21

Fig.1 The structure of QT.

Liyan Zhao, et al., Fig. 1

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

23

Fig.3 TEM image of QT loaded P123/TPGS mixed micelles (×19,0000).

Liyan Zhao, et al., Fig. 3

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

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

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 profiles of QT from QT-P123(▲), QT-P/T(■) and the propylene glycol

solution (◆) in 0.5% Tween 80 solution at 37 ºC. Each point represents average

± SD (n = 3).

Liyan Zhao, et al., Fig. 7

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 (μg/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 (μg/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 (μg/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 (μg/ml)

Cel

l via

bilit

y

empty QT-P/T

empty QT-P123