Journal of Colloid and Interface Science 434 (2014) 40–47

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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis

Preparation, optimization, characterization and cytotoxicity in vitro of Baicalin-loaded mixed micelles Haiqun Zhang a, Lili Zhao a, Lianjun Chu b, Xu Han c, Guangxi Zhai a,⇑ a

Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan 250012, China College of Medicine and Nursing, Dezhou University, Dezhou 253023, China c College of Chemistry, Shandong University, Jinan 250100, China b

a r t i c l e

i n f o

Article history: Received 18 May 2014 Accepted 30 July 2014 Available online 8 August 2014 Keywords: Baicalin Pluronic P123 copolymer Sodium taurocholate Mixed micelles Entrapment efficacy

a b s t r a c t The aim of this study was to develop a Baicalin (BC)-loaded mixed micelle delivery system (BC–ST–P123– MMs) with sodium taurocholate (ST) and pluronic P123 block copolymer (P123) as carrier materials to improve the solubility of BC, a poorly soluble drug. In this study, the mixed micelle system was prepared using the method of thin-film dispersion and then optimized by the homogeneous design–response surface methodology with the entrapment efficiency and drug loading as indexes. The average size and the zeta potential of the BC–ST–P123–MMs were 15.60 nm and 5.26 mV, respectively. Drug loading (DL, 16.94%) and entrapment efficiency (EE, 90.67%) contributed to high solubility (10.20 mg/mL) of BC in water. The optimized BC–ST–P123–MMs appeared spherical with obvious core–shell structure and well dispersed without aggregation and adhesion under TEM. In addition, DSC result indicated that BC had been wrapped in BC–ST–P123–MMs and crystalline state of BC was changed. The release result in vitro showed that BC–ST–P123–MMs presented sustained release behavior compared to control group. The IC50 value of BC–ST–P123–MMs (46.18 lg/mL) was lower than that of BC solution (67.14 lg/mL) on Hep G2 cell lines. Cellular uptake tests illustrated that the ST–P123–MMs system as carrier could significantly enhance the uptake of drugs by tumor cells. The results demonstrated that the BC-loaded mixed micelles could improve solubility of BC and exhibited great potential for delivering drug into cancer cells. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Baicalin (BC) (Fig. 1), a kind of flavonoids, is the main effective component of radix scutellariae and has widespread biological activities and pharmacological activities such as antibacterial, anti-inflammatory, antioxidant, diuresis, calm, step-down, immunosuppression and spasmolysis functions [1–3]. In the recent years, strong effect on cancer and HIV of BC was found [4–6]. In spite of plenty of superiorities, the low water-solubility of BC brought many defects such as weak stability in solution, poor absorption and low bioavailability [2,7]. A series of BC-loaded formulations have been developed including gelling system [8], solid lipid nanoparticles [9], phospholipid complex [10], coprecipitate [11], nanoemulsion [12], and solid nanocrystals [13]. Up to now, no micelle formulation has been reported for improving the solubility and enhancing treatment effect of BC. Considering these

⇑ Corresponding author. Address: Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua Xilu, Jinan 250012, China. E-mail address: [email protected] (G. Zhai). http://dx.doi.org/10.1016/j.jcis.2014.07.045 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.

advantages of micelle such as high solubilization capacity, small particle size, hydrophilic shell, long circulation and passive targeting ability, we designed and developed BC-loaded micelles by searching appropriate carrier materials and then optimized the preparation technology [14–19]. Based on the theory of synergistic effect, the mixed micelle system formed by two or more materials exhibited preferable characteristics than conventional micelle composed of single carrier material and attracted increasing attention in the recent years [20,21]. Numerous investigators successfully improved the solubility and bioavailability of hydrophobic drugs by mixed micelles strategy, such as P123 and F127 , P123 and C12EO6, PEG–PE and TPGS, PEG–PE and vitamin E and P123 and F68 [22–29]. Bile salts, as anionic surface active agents, have the strong solubilization and biocompatibility, which play an important role in many physiological and biological systems known as the ‘physiological detergent’ [30–32]. Sodium taurocholate (ST), a conjugate combined by the carboxyl of bile acid and amino of taurine, is widely used in pharmacy on account of its evident effects of antitussive, expectorant, antiasthmatic and surface-active advantage in particular [33,34]. Block copolymer poly(ethylene oxide)-b-

H. Zhang et al. / Journal of Colloid and Interface Science 434 (2014) 40–47

Fig. 1. The chemical structure of Baicalin.

poly(propylene oxide)-b-poly(ethylene oxide) (PEO–PPO–PEO, or pluronic 123 copolymer, or P123) is used as carrier material for solubilization and delivery of drug in view of its non-toxicity, nonimmunogenicity and biocompatibility [35]. Moreover, the high proportion of hydrophobic block could account for ideal drug loading ability of P123 [36–38]. As amphipathic molecule, both ST and P123 could self-assemble to form micelles in aqueous solution [39]. The mixed micelle system composed of ST and P123 should incorporate water-insoluble drug into the hydrophobic core away from outer environment to enhance the solubility, stability and bioavailability of the drugs. Based on these considerations, the collaboration of ST and P123 would be suitable as carrier materials for loading and delivering BC. In the study, BC–ST–P123–MMs system was prepared by the thin-film dispersion method and optimized utilizing the homogeneous design–response surface methodology. Physicochemical properties of this developed formulation, such as surface morphology, size distribution, zeta potential, critical micelle concentration, loading capacity, entrapment efficiency, phase state and in vitro release were characterized. Cell uptake and cytotoxicity in vitro were tested to further confirm the superiority of BC–ST–P123– MMs in enhancing the therapeutic effect of model drug. 2. Materials and methods

41

under reduced pressure to obtain a mixed film composed of drug and carrier materials. The mixture film was further dried in vacuum overnight to get rid of redundant ethanol. After that, the dried film was hydrated with deionized water and then dispersed via vortex and agitation. The micellar solution was centrifuged at 10,000 rpm for 10 min to remove unloaded hydrophobic drug by centrifuge H1650-W (Xiangyi, Hunan). The supernatant containing BC–ST–P123–MMs was collected. In addition, blank ST–P123–MMs were prepared via the same way just without BC. For DSC and TGA assays, mannitol, BC suspension, blank ST– P123–MMs solution and BC–ST–P123–MMs solution were lyophilized to obtain mannitol, BC, ST–P123–MMs and BC–ST–P123– MMs freeze-dried powders. Mannitol as stabilizer was fed into these four kinds of solvents before lyophilization at equal quantity. The freeze-dried powders of BC and ST–P123–MMs were blended with each other to get BC + ST–P123 physical mixture. The prepared lyophilized powders were all preserved in cold storage for the determination. To study the uptake of ST–P123–MMs by cancer cells, the coumarin-6 (fluorescent material) loaded mixed micelles were prepared by the same way. 2.3. Optimization of the preparation technology A series of single-factor experiments were carried out such as the amount of P123, ST, BC and the volume of hydrating solution, evaporated temperature and time, hydrated temperature and time. The results of single-factor experiments showed that the amount of P123, ST, BC and hydration volume of deionized water had a significant impact on drug loading capacity and encapsulation efficiency of mixed micelles. So the four factors were optimized with the homogeneous design–response surface method. The design code values of the amount of P123, ST, BC and deionized water volume of hydration are listed in Table 1. 2.4. Physicochemical characterization of Baicalin-loaded micelles 2.4.1. Particle size and zeta potential Particle size and zeta potential of BC–ST–P123–MMs were measured using Delsa Nano (Beckman coulter) at 25 °C. Each sample was diluted with distilled water to obtain an appropriate light intensity for measurement and all samples were measured in triplicate (n = 3).

2.1. Materials BC was purchased from D&B (Shanghai, China). ST was purchased from Solarbio (Beijing, China). P123 (Mn  5800) and coumarin-6 were purchased from Sigma–Aldrich (USA). 3-(4,5Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Amresco (USA). RPMI 1640 were purchased from Thermo Fisher (Beijing, China). Trypsin–EDTA was purchased from Gibco (USA). Fetal bovine serum (FBS) was purchased Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Zhejiang, China). Human hepatocellular carcinoma cell lines (Hep G2) and human cervical carcinoma cell lines (Hela) were donated by Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University. Other chemicals in all studies were of analytical grade. 2.2. Preparation of Baicalin-loaded mixed micelles BC–ST–P123–MMs were prepared by thin-film dispersion method [40]. Briefly, BC, ST and P123 of various concentrations and proportions were dissolved completely in ethanol by ultrasound at room temperature. Then, the ethanol was evaporated

2.4.2. Morphology observation The morphology of the final optimum formulation was observed under transmission electron microscopy (TEM). A drop of BC–ST–P123–MMs solution was placed on a copper grid and stained with phosphotungstic acid solution (2%, w/v) for 10 s. After the excess solution was removed, the sample was dried in air and the micelle morphology was observed under TEM. 2.5. Entrapment efficiency (EE) and drug loading (DL) The loading amount of BC in BC–ST–P123–MMs was determined by UV–Vis spectrophotometer at 317 nm. The developed micelle formulation was diluted to an appropriate concentration with methanol before determination. Drug loading (DL%) and entrapment efficiency (EE%) were calculated by the following equations [41]:

DL% ¼

weight of BC in micelles  100 weight of BC in micelles þ weight of feeding P123 and ST

ð1Þ EE% ¼

weight of BC in micelles  100 weight of feeding BC

ð2Þ

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H. Zhang et al. / Journal of Colloid and Interface Science 434 (2014) 40–47

Table 1 The design values and results of uniform design experiment. No.

1 2 3 4 5 6 7 8 9

Values of variables

Results

Amount of P123 (mg)

Amount of ST (mg)

Amount of BC (mg)

Volume of hydration solution (mL)

EE (%)

DL (%)

60 60 60 90 90 90 120 120 120

60 90 90 120 60 60 90 120 120

30 40 20 40 20 30 20 30 40

5 4 3 3 5 4 4 3 5

70.40 72.62 70.75 71.88 75.06 85.20 84.15 73.00 83.56

14.08 15.29 8.32 11.50 8.83 14.20 7.32 8.11 11.94

2.6. Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) DSC/TGA analyses of mannitol, BC, ST–P123–MMs, BC + ST– P123 physical mixture and BC–ST–P123–MMs freeze-dried powders were performed using a Diamond DSC (PerkinElmer, Waltham, MA, USA) equipped with an intercooler [11]. Calibration for temperature and heat of fusion was performed with indium and tin as reference materials. The samples were evaluated in open aluminum pans and scanned under a nitrogen purge with a heating rate of 10 °C/min from 50 °C to 275 °C. 2.7. In vitro release study The release property of BC–ST–P123–MMs was tested using the dialysis method [42]. Briefly, 300 lL of BC–ST–P123–MMs as experimental group and BC solution (dissolved in propylene glycol) at the same concentration as control group were added to dialysis bags with cut-off MW of 8000–14,000 (Solarbio, Beijing, China). The dialysis bags were placed into 100 mL release medium of PBS buffer (pH 7.4). The completely released BC concentration was 40.80 lg/mL under the leakage condition. Then dialysis bags were shaken at a speed of 100 rpm at 37 °C. 1 mL of release medium was taken out and the equal volume of fresh release medium was replenished quickly to maintain the constant volume (100 mL) at the predetermined intervals. Samples were filtered with 0.22 lm filters and then determined by UV–Vis spectrophotometer at 317 nm. All results were obtained in triplicate measurements. 2.8. In vitro cell toxicity assays Cell toxicity test of BC loaded mixed micelles system was performed with MTT method. Hep G2 cell lines, which were sensitive to BC, were selected for the toxicity experiment [4]. Hep G2 cells in logarithmic phase were seeded in 96-well plates with cell densities being approximately 5  103 cells per well. When cells reached 90– 95% confluence, BC solution (0.5% DMSO), blank ST–P123–MMs and BC–ST–P123–MMs were added into the cell lines. The crude BC solution and BC–ST–P123–MMs groups were set at same BC concentration. In addition, blank ST–P123–MMs group were diluted at equal carrier materials concentration in accordance with BC–ST–P123– MMs group. After incubation for 48 h, medium was removed and fresh medium with 20 ll of MTT solution (5 mg/mL) was added. After 4 h, the medium was withdrawn and 200 ll of DMSO was used to dissolve the formazan crystals produced by living cells. MTT assay was measured by multiwell scanning spectrophotometer Model 680 (Bio-Rad, USA) at 570 nm and 630 nm [43]. The computational formula of cell inhibition was as follows (each concentration was set up in six replicates):

Cell inhibition% ¼

  Absorbanceexperimental 1  100% Absorbancecontrol

ð3Þ

Absorbanceexperimental stands for the absorbance of cells interacted with micelles or drugs loaded micelles. Absorbancecontrol represents the absorbance of cells cultured without micelles and drugs. 2.9. In vitro cellular uptake assays Coumarin-6 was generally used as a fluorescence probe incorporated into nano-sized particles for observation of cellular uptake [44]. In view of water-insoluble property of BC, coumarin-6 was selected as a similar substitution of BC and incorporated into ST– P123–MMs. Hep G2 and Hela cells were selected for cell uptake experiment. The two cells in logarithmic phase were seeded in 12-well plates with densities being approximately 1  105 cells per well and incubated until they reached 90–95% aggregation extent. Then the medium was replaced with fresh medium containing coumarin-6 solution and coumarin-6 loaded ST–P123– MMs. After 2 h and 4 h incubation, cells were washed with cold PBS (pH 7.4) three times to eliminate residual coumarin-6 outside cells. Fluorescence intensities of coumarin-6 inside two cells were observed under fluorescence microscope (Olympus, JP) qualitatively. Then the two cells were trypsinized and collected and cleaned with cold PBS. Cells were suspended in PBS and then cell uptake rate was determined quantitatively by flow cytometry (FCM) (BD, USA). 2.10. Statistical analysis The statistical significance of differences among more than two groups was determined by one-way ANOVA. A value of P < 0.05 was considered to be significant. 3. Results and discussion 3.1. Optimization of the preparation technology According to previous study about micellization temperature of P123 in mixed micelles and the aim to maintain persistent stability of the mixed micelles in vivo, the vaporization temperature and hydration temperature in the process of ST–P123–MMs were set at 37 °C [24,25]. In addition, a series of single-factor experiments were performed to select unilateral control factors which played important influences on preparation technology with DL and EE as indexes. The results indicated that DL and EE were obviously affected by the amount of P123, ST, BC and hydration volume of distilled water. Then homogeneous design (HD) with the amount of P123, ST, BC and hydration volume of distilled water as control factors was conducted for further optimization. The four variables were optimized through HD at three experimental levels. Amount of ST was set as X1 ranging from 60 to 120 mg, amount of P123 as X2 in the range from 60 to 120 mg, the amount of BC as X3 from 20

H. Zhang et al. / Journal of Colloid and Interface Science 434 (2014) 40–47

to 40 mg and hydration volume of distilled water as X4 from 3 to 5 mL. The experimental design and results are listed in Table 1. The results were dealt with SPSS 17.0 statistical software making multivariate nonlinear regression. The quadratic polynomial equation was obtained as follows,

Y EE % ¼ 0:279  2:702  106 X 21  1:115  105 5X 22  0:004X 24 þ 3:666  105 X 1 X 3 þ 0:001X 3 X 4 The equation could demonstrate that the four dependent variables had high correlation with encapsulation efficiency of the developed formulations (R > 0.95, P < 0.05). Then response surface plots (Fig. 2a and b) and corresponding contour maps (Fig. 2c and d) were described based on quadratic polynomial equations by Origin 8.5 software. The optimal area of entrapment efficiency could be obtained from the areas of surface plots and contour maps. Besides, considering favorable stability and appropriate particle size of the micelle solution, several additional experiments were carried out in the range of optimal area to find out the optimal formulation. Based on the optimization and further screening experiments, the final formulation was as follows, X1 = 100 mg, X2 = 100 mg, X3 = 45 mg, X4 = 4 mL. The solubility of BC in the developed mixed micelle system was increased to 10.20 mg/mL, which was notably higher than that of coarse BC in water (53.51 lg/mL) [13]. The high DL and EE were attributed to the hydrophobic interaction of lipophilic drugs and hydrophobic core of micelles [29].

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3.2. Particle size, zeta potential, morphology of mixed micelles The mean size of BC–ST–P123–MMs was 15.6 ± 0.3 nm with a polydispersity index of 0.19 ± 0.01 (Fig. 3a), and the average zeta potential measured was 5.36 ± 0.19 mV. Particle size contributed to the anticipant tissue distribution and passive targeting ability of micelles in vivo [45]. It was well known that nano-particles with the size ranging from 10 nm to 100 nm could avoid being filtered out by kidneys or absorbed and metabolized by liver [46,47]. Therefore, this developed BC–ST–P123–MMs formulation with particle size of 15.6 nm could avoid rapid metabolism and elimination and guarantee the stability and long circulation time. Besides, the nano-sized micelles accounted for the enhanced permeability and retention (EPR) effect, i.e., passive targeting ability. The electrostatic repulsion, steric hindrance and hydrophilic interactions between hydrophilic chains shell of mixed micelles system could prevent the aggregation of the micelles and provide important effects on stability of the colloid system [27]. Under TEM, the mixed micelles presented perfectly spheroidal shape and obvious core–shell structure with no conglutination (Fig. 3b). With respect to the surface morphology, BC–ST–P123– MMs aqueous solution was clear and transparent, while the BC suspension dispersing in water at equal mass with BC–ST–P123– MMs group appeared turbid, non-transparent and insoluble state (Fig. 3c). Based on the amphiphilic property and block structure of carrier material [38], it was deduced that P123 with larger hydrophobic chain area formed the core, and ST with larger hydro-

Fig. 2. Response surface plots of X1  X2 (a) and X3  X4 (b), and response surface contour plots of X1  X2 (c) and X3  X4 (d).

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c

b

b

a

ST P123

BC

d

Fig. 3. The particle size distribution (a), the morphology image of BC–ST–P123–MMs under TEM (b), external photos of BC–ST–P123–MMs (c-i) and BC suspension at the same concentration (c-ii) and schematic illustration of the micelle formation of BC–ST–P123–MMs (d).

in vitro release

DSC



1.5



0.5

BC

mW/mg



BC+ST-P123 physical mixture

-1.5 -2.5

BC-ST-P123-MMs

-3.5 -4.5 mannitol

-5.5 -6.5 -7.5 50

    

BC Solution

 BC-ST-P123-MMs

 80

110

140

170

200

230

260

Temperature °C TGA

105

BC-ST-P123-MMs

95















Time (h) mannitol

Fig. 5. Profiles of the cumulative release percentage of BC from mixed micelles (j) and propylene glycol solution () in PBS (pH = 7.4).

ST-P123-MMs

BC+ST-P123 physical mixture

90

 

100

Weight %

Cumulative Release (%)

ST-P123-MMs

-0.5

3.3. DSC and TGA analyses

85 BC

80 75 70 65 50

80

110

140

170

200

230

260

Temperature °C Fig. 4. DSC thermographs and TGA atlas of mannitol, BC, ST–P123–MMs, BC + ST– P123 physical mixture and BC–ST–P123–MMs.

philic area inserted to hydrophilic chains of P123 and distributed around micelle core as outer shell [40]. The graphical representation of the structure of the mixed micelles is shown in Fig. 3d.

DSC and TGA could be applied for detecting the change of crystalline state after BC was encapsulated in BC–ST–P123–MMs [48]. As shown in DSC diagram (Fig. 4), there was a sharp decalescence peak at 199.77 °C as melting point in BC group. Mannitol showed no interference near this decalescence peak as stabilizer. So mannitol existing in ST–P123–MMs, BC + ST–P123 physical mixture and BC–ST–P123–MMs freeze-dried powders had no influence on analysis result. This diagram showed that the melting peak of BC could also be found in the BC + ST–P123 physical mixture, revealing that there was no phase change of BC. Melting peak was not found in ST–P123–MMs and BC–ST–P123–MMs, indicating that the crystalline state of BC in BC–ST–P123–MMs was disappeared. The same results were obtained from TGA figure (Fig. 4). Mannitol also had no interference on TGA analysis. The BC–P123–ST–MMs appeared the same weightlessness trend as ST–P123–MMs, while

H. Zhang et al. / Journal of Colloid and Interface Science 434 (2014) 40–47

trated that the phase state of BC had changed and the drug was highly dispersed in the ST–P123–MMs system.

Cells toxicity assay 100

Cell inhibition rate (%)

45

90 80

3.4. In vitro release

70 60 50 40 30 20 10 0

0

50

100

150

200

Concentration (µg/ml)

Fig. 5 exhibited the release profiles of BC from mixed micelles and propylene glycol solution, respectively. 15.26% of incorporated BC was released from micelles within 2 h, which showed slight burst release. For the control group, 92.87% of BC from propylene glycol solution was released rapidly within 8 h. However, only 55.78% of BC was released from the micellar formulation during the same time. After 48 h, 16.97% of the incorporated drug was still detained in mixed micelles. The slow release behavior confirmed the stability of the BC encapsulated in inner core of mixed micelles for long circulation time.

Fig. 6. Cell inhibition rates of free drug solution (j), blank ST–P123–MMs (N) and BC–ST–P123–MMs () with different concentrate on Hep G2 cells for 48 h.

3.5. Cells toxicity assay

the result was different with BC and BC + ST–P123 physical mixtures obviously. The experimental results of DSC and TGA illus-

Cells toxicity assay was performed on Hep G2 cell lines [49]. Cells inhibition results of crude BC solution, blank ST–P123–MMs

Hela

Hep G2

Control-2h

Control-4h

Micelles-2h

Micelles-4h Fig. 7. Fluorescent images of Hela and Hep G2 cells after 2 h and 4 h incubation with free coumarin-6 solution and coumarin-6-loaded ST–P123–MMs solution.

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H. Zhang et al. / Journal of Colloid and Interface Science 434 (2014) 40–47

A Coumarin-6-loaded ST-P123-MMs

Coumarin-6 solution

Control

B Control

Coumarin-6-loaded ST-P123-MMs

Coumarin-6 solution

Fig. 8. Flow cytometry test results of Hela (A) and Hep G2 cell lines (B) incubation with coumarin-6 solution and coumarin-6-loaded ST–P123–MMs for 2 h.

and BC–ST–P123–MMs at different concentrations on Hep G2 cells were shown in Fig. 6. The results showed that the IC50 values of BC–ST–P123–MMs and BC solution on Hep G2 cells were 46.18 lg/mL and 67.14 lg/mL, respectively. IC50, the half maximal inhibitory concentration, represents the drug concentration at which 50% cancer cells growth was inhibited. Lower IC50 value indicated higher cytotoxicity of drug. So, it was concluded that the developed BC–ST–P123–MMs had higher cytotoxicity and therapeutic effect than BC solution at the same amount of drug. 3.6. Cellular uptake tests The cellular uptakes of coumarin-6-ST–P123 mixed micelles and coumarin-6 solution were conducted on Hela and Hep G2 cell lines. The coumarin-6 solution as control group was set at equal concentration of coumarin-6 (10 lg/mL). Fluorescence microscope was used to visualize the cellular uptake ability of coumarin-6-ST– P123 mixed micelles and coumarin-6 solution. The fluorescent images of Hela and Hep G2 cells incubated with coumarin-6 solution and coumarin-6 loaded ST–P123 mixed micelles are shown in Fig. 7. It can be observed that the coumarin-6 loaded ST–P123 mixed micelles group showed stronger fluorescent signals at 2 h and 4 h than the coumarin-6 solution group. Stronger fluorescent signals indicated higher internalization of coumarin-6. The results revealed that ST–P123–MMs could significantly enhance the cellular uptake of coumarin-6. To compare cellular uptake ability of coumarin-6-ST–P123– MMs and the coumarin-6 solution quantitatively, the test was conducted by FCM technique. As shown in Fig. 8, the results also demonstrated that both Hela and Hep G2 cells had higher cellular absorbed quantity on coumarin-6-ST–P123–MMs than that on

the coumarin-6 solution. In other words, this developed mixed micelles system could deliver more drugs into cancer cells and enhance therapeutic effect of crude drug. All these results confirmed that the developed mixed micelles exhibited great cellular uptake efficiency on both Hela and Hep G2 cells and enhanced cytotoxicity on Hep G2 cells [30,31,36].

4. Conclusion Baicalin (BC) is regarded as a promising agent against various diseases with wide spectrum of biological activities. However, the application of BC was hampered owing to the low solubility in water. In this study, BC-loaded P123–ST–MMs system was prepared by thin film dispersed method and realized high solubility of BC in aqueous solution (10.20 mg/mL). This developed formulation showed high loading capacity (16.94%) and entrapment efficiency (90.67%) and sustained release property. DSC and TGA measurements revealed that the crystalline state of BC in BC–ST–P123– MMs was disappeared and the phase state changed, which proved that BC had been incorporated into ST–P123–MMs. The cell assays further confirmed higher cellular internalization ability of coumarin-6-ST–P123–MMs and greater cytotoxicity of BC–ST–P123– MMs than corresponding control groups. All these results proved that the developed ST–P123–MMs system was a potential carrier system for delivery of BC. Acknowledgments This work was partly supported by the Natural Science Foundation of Shandong Province, China (No. ZR2011HM026) and the

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