http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2013.873501
ORIGINAL ARTICLE
Curcumin-loaded mixed micelles: preparation, optimization, physicochemical properties and cytotoxicity in vitro Yuwei Duan1, Juan Wang2, Xiaoye Yang1, Hongliang Du1, Yanwei Xi1, and Guangxi Zhai1 Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan, China and 2Department of Pharmacy, Jinan Maternity and Child Care Hospital, Jinan, China
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Abstract
Keywords
Although curcumin (CUR) can inhibit proliferation and induce apoptosis of tumors, the poor water solubility restricted its clinical application. The aim of this study was to improve the aqueous solubility of CUR and make more favorable changes to bioactivity by preparing curcumin-loaded phospholipid-sodium deoxycholate-mixed micelles (CUR-PC-SDC-MMs). CUR-PC-SDC-MMs were prepared by the thin-film dispersion method. Based on the results of single factor exploration, the preparation technology was optimized using the central composite design-response surface methodology with drug loading and entrapment efficiency (EE%) as indicators. The images of transmission electron microscopy showed that the optimized CUR-PC-SDC-MMs were spherical and well dispersed. The average size of the mixed micelles was 66.5 nm, the zeta potential was about 26.96 mV and critical micelle concentration was 0.0087 g/l. CUR was encapsulated in PC-SDC-MMs with loading capacity of 13.12%, EE% of 87.58%, and the solubility of CUR in water was 3.14 mg/ml. The release results in vitro showed that the mixed micelles presented sustained release behavior compared to the propylene glycol solution of CUR. The IC50 values of CUR-loaded micelles and free drug in human breast carcinoma cell lines were 4.10 mg/ml and 6.93 mg/ml, respectively. It could be concluded from the above results that the CUR-PC-SDC-MMs system might serve as a promising nanocarrier to improve the solubility and bioactivity of CUR.
Curcumin, cytotoxicity, drug delivery system, phospholipid-sodium deoxycholate, solubility
Introduction The mixed micellar system formed by different surface active agents mixed with each other, because of the better performance than single surfactant, has attracted increasing attention in recent years (Zhu et al., 2010). Bile salts and phospholipids (PC) as anionic and amphoteric surfactants are both amphipathic molecules, which can show the characteristic of self assembly in the aqueous solution (Hernell et al., 1990; Sugioka & Moroi, 1999; Su & Wang, 2004). For organism, bile salts, secreted by liver and stored in the gall bladder, could form a kind of stable mixed micellar system with PCs, glycerides and fatty acids during lipid digestion in the small intestine (Yu et al., 2010; Duan et al., 2011). The bile salt/PC micelles can package cholesterol inside the hydrophobic core, and the solubility, stability and bioavailability of poorly water-soluble drugs could be enhanced in the same way (Dangi et al., 1995; Wiedmann & Kamel, 2002; Wiedmann et al., 2002; Garidel et al., 2007). Meanwhile, water-soluble drugs would insert into the hydrophilic palisade
Address for correspondence: Guangxi Zhai, PhD, Department of Pharmaceutics, College of Pharmacy, Shandong University, 44 Wenhua Xilu, Jinan 250012, China. Tel: (86) 531-88382015. Fax: (86) 531-88382731. Email: [email protected]
History Received 13 October 2013 Revised 29 November 2013 Accepted 5 December 2013
layer of micelles to separate from the surrounding unavailable environment. Many investigators have successfully improved the solubility and bioavailability of insoluble drugs in this way, such as fat-soluble vitamins, paclitaxel, lorazepam and diazepam (Chen et al., 2001). Besides the advantages above, bile salts, PCs and their degradation products, as components existing in organism, could be absorbed by human body completely. As a result, the combination of bile salts and PCs, which raises the safety of preparation, is considered as biocompatible injection solvent (Mattila & Suistomaa, 1984) and a promising delivery system for anticancer drugs. Curcumin (CUR), a yellow acidic polyphenol compound, is an effective active ingredient extracted from the rhizome of the perennial herb Curcuma longa (Gandapu et al., 2011). It has been used in traditional medicine for many years with a wide range of pharmacological actions (Shishodia et al., 2005), such as anti-inflammatory, antioxidant, anti-microbial and anti-tumoral effects (Srimal & Dhawan, 1973; Sharma, 1976; Kuttan et al., 1985; Mahady et al., 2001). CUR is capable to defend against cancer in vitro and in vivo through multiple mechanisms. It can prevent the proliferation and induce apoptosis of various tumor cells (Aggarwal et al., 2003). High doses of CUR (12 g/d) given to clinical models are better tolerated, which shows CUR is safe for organism (Hsu & Cheng, 2007). However, whether oral administration or injection, the bioavailability of CUR is poor with
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properties of insoluble in water, easily metabolized and fast systemic eliminated, which has become the main block of the clinical application (Anand et al., 2007). Improving the solubility, stability and bioavailability is of significant importance to the wide usage of CUR as therapeutic agent. Until now, a series of dosage forms for CUR have been developed including nanosuspension, nanoparticles, nanospheres and inclusion complex, etc. Based on the shortcomings of CUR and the superiority of bile salt/PC-mixed micelles, this article reports a new injectionable micelle formulation to enhance the solubility and bioactivity of CUR. Curcumin-loaded phospholipid-sodium deoxycholate mixed micelles (CURSDC-PC-MMs) were prepared by the thin-film dispersion method. Then the preparation was optimized with the central composite design (CCD)-response surface method. In addition, the physicochemical properties and the cell toxicity of CUR-PC-SDC-MMs in vitro were investigated.
Materials and methods Materials CUR and SDC were purchased from Sigma-Aldrich (St. Louis, MO). Soybean lecithin (PC) was purchased from Shang Hai Tai Wei Pharmaceutical Co., Ltd. (Shanghai, China). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), trypsin and ethylenediaminetetraacetic acid were purchased from Amresco (Solon, OH). RPMI 1640 was purchased from Thermo Fisher (Beijing, China). Fetal bovine serum (FBS) was purchased from Tian Jin Hao Yang Biological Manufacture Co., Ltd. (Tianjin, China). Human breast carcinoma cell lines (MCF-7) were donated by Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University. All other chemicals were of analytical grade.
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Study on physicochemical properties Particle size and zeta potential Particle size and zeta potential of CUR-PC-SDC-MMs were measured using DelsaNano-based-Beckman coulter at 25 C. Each sample was measured in triplicate. Micro-morphology The morphology of optimum CUR-PC-SDC-MMs was observed by the transmission electron microscopy (TEM). A drop of the formulation was placed on a copper grid and stained with phosphotungstic acid solution (2%, w/v) for 15 s. Then, the excess solution was absorbed, and the sample was dried in air and examined under TEM. Drug loading and EE% The content of CUR encapsulated in PC-SDC-MMs was determined by UV-Vis spectrophotometer at 426 nm. The prepared micellar solution was diluted to a certain concentration with anhydrous ethanol before determination. The drug loading (DL%) and entrapment efficiency (EE%) were calculated by the following equations (Jansson et al., 2004): WCUR DL% ¼ 100% ð1Þ Wmicelle WCUR 100% ð2Þ WCUR0 where WCUR, the weight of drug in micelles; WCUR’, the weight of feeding drug; and Wmicelle, the total weight of feeding PC, SDC and drug in micelles. It is reported that the solubility of CUR in water was 11 ng/ml (Cui et al., 2009), much smaller compared with loading content of CUR inside micelles, so DL% and EE% were calculated ignoring the content of CUR in water (Chen et al., 2001). EE% ¼
Preparation of CUR-PC-SDC-MMs
Critical micelle concentration
CUR-PC-SDC-MMs were formed through thin film dispersed method. Briefly, CUR, PC and SDC (with different concentrations and proportions) were dissolved in anhydrous ethanol by stirring at room temperature. Then, the solution was evaporated at 40 C under reduced pressure to obtain a thin film of drug-carrier materials, and the film was further dried in vacuum drying oven over night to remove redundant organic solvent. After that, the film was hydrated in 5 ml PBS and dispersed for 15 min via ultrasound. The micellar suspension was centrifuged at 12 000rpm for 5 min in order to separate unloaded drugs, and the supernatant was collected, which contained CUR-PC-SDC-MMs. The empty micelles were prepared in the same way just without CUR.
The critical micelle concentration (CMC) of PC-SDC in the PBS (pH 7.4) was determined with pyrene fluorescence probe method using F-2500 fluorescence spectrophotometer. The prepared blank micelles were diluted to a series of concentrations ranging from 1 103 to 2 g/l. Pyrene dissolved in acetone was added to a few of amber glass vials, and the acetone was evaporated with continuous flow of nitrogen gas (Chen et al., 2001). Micellar solutions with different concentrations were added into the amber glass vials with a final pyrene concentration of 2 107 mol/l, and the solution was mixed uniformly. Then the mixtures were incubated for balance in the dark for 24 h at room temperature prior to measurement. Fluorescence emission spectrum was scanned from 350 to 450 nm with the rate at 1200 nm/min, at an excitation wavelength of 334 nm. The slits of excitation and emission were 5 nm and 2.5 nm, respectively. The fluorescence intensity at wavelength 372 and 383 nm was recorded, respectively, and the ratio between I372 and I383 would be alternated with pyrene moving into the interior of micelles. All experiments were performed in triplicate.
Optimization of the preparation technology In order to optimize the preparation technology, the CCDresponse surface method was used based on single-factor experiments such as the amount of CUR added, the mass ratio of PC to SDC, the volume of hydration solution, hydration temperature and time.
Curcumin-loaded mixed micelles
DOI: 10.3109/10717544.2013.873501
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In vitro release of CUR The release behavior of CUR from micellar system was studied with physiological saline containing 1% Tween 80 (w/v) as release medium using dialysis method. Under the premise of sink condition, an appropriate volume of CUR-PC-SDC-MMs and CUR solution (dissolved in propylene glycol) as control, with an equal content of CUR, was placed into a pre-swollen dialysis membrane bag. The dialysis bag was tied and placed into an Erlenmeyer flask containing 50 ml of release medium. The temperature and stirring speed were set at 37 C and 120 r/min separately. At each pre-set time point, 1 ml of the medium was withdrawn from the flask and 1 ml of fresh medium was added into the flask. The samples taken out were filtered through 0.22 mm filter membrane, and the filter liquor was measured by high-performance liquid chromatography (HPLC) at 426 nm. A Hypersil BDS C18 reserved phase analytical column (5 mm, 4.6 mm 250 mm) was applied in HPLC analyses. The mobile phase consisted of methanol/ water containing 3.6% glacial acetic acid (70:30) was pumped at the rate of 1.0 ml/min. The column temperature was set at 25 C. The cumulative release percentage of CUR was calculated. Every release experiment was repeated three times. Cell toxicity assays MCF-7 cells were adopted to evaluate the in vitro toxicity of CUR-PC-SDC-MMs with the MTT method. Cells were cultured in the RPMI-1640 medium supplemented with 10% FBS. All the cells were grown at 37 C in 5% CO2 atmosphere in a humidified incubator, and subcultured once every two days. MCF-7 cell lines were seeded in 96-well culture plates at the density of 5 103 per well. After 24 h of incubation, cells were handled with CUR-PC-SDC-MMs of different concentrations, blank micelles and CUR solution dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was below 0.2% according to references (Zhao et al., 2012), and the concentrations of treatment agents ranged from 0.75 mg/ml to 24 mg/ml. After incubation for 48 h, medium in well was withdrawn and phosphate buffer saline was added to wash the well. Next, each well was
replenished with fresh culture medium with 20 ml of MTT (5 mg/ml) in it. The culture plates were incubated for 4 h at 37 C and centrifuged for 10 min at 3000 rpm/min. Subsequently, the medium was moved out and 150 ml of DMSO was added to dissolve the formazan crystals produced inside cells. Absorbance of cells were measured by multiwell scanning spectrophotometer Model 680 (Bio-Rad, Hercules, CA) at 570 nm and 630 nm (Zhao et al., 2012). Each concentration was set up in six replicates, and experiment was measured in triplicate. The formula of cell inhibition percentage was as follows: Absorbanceexperimental Cell Inhibition % ¼ 1 100% Absorbancecontrol where Absorbanceexperimental, the absorbance of cells interacted with micelles or drug; Absorbancecontrol, the absorbance of cells cultured with no micelles or drug.
Results Optimization of the preparation technology Taking DL% and EE% as index, a series of single-factor experiments were performed to select the control factors, which influenced preparation technology significantly. The results indicated that DL% and EE% had obvious changes along with varying amount of CUR and the mass ratio of PC to SDC. Hence, the two factors were conducted as variables for optimization through CCD at five experimental levels. Amount of CUR was as X1 ranging from 1.25 to 20 mg, and the mass ratio of PC to SDC was as X2 in the range from 0 to 2.333. The experimental design and results were shown in Table 1. The data was dealt with SPSS 17.0 (SPSS Inc., Chicago, IL) statistical software making multivariate nonlinear regression. The quadratic polynomial equations were obtained as follows: YDL% ¼ 1:127 þ 1:018X1 þ 2:305X2 0:011X21 0:790X22 0:126X1 X2 YEE% ¼ 86:879 þ 19:200X2 0:799X1 X2 8:243X22
Table 1. Central composite design with code values and observed values. Code values of variables
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Actual values of variables
Observed
No.
X1
X2
Amount of CUR
the mass ratio of PC and SDC
DL (%)
EE (%)
1 2 3 4 5 6 7 8 9 10 11 12 13
1 1 1 1 1.414 1.414 0 0 0 0 0 0 0
1 1 1 1 0 0 1.414 1.414 0 0 0 0 0
17.255 17.255 3.995 3.995 20.000 1.250 10.625 10.625 10.625 10.625 10.625 10.625 10.625
1.991 0.342 1.991 0.342 1.167 1.167 2.333 0 1.167 1.167 1.167 1.167 1.167
10.38 13.10 3.51 3.47 13.87 1.26 6.31 8.52 8.55 8.53 8.50 8.53 8.65
66.53 87.19 91.01 92.15 81.35 94.48 63.10 87.44 87.41 87.03 86.84 87.22 89.38
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Figure 1. Response surface plots and response surface contour plots based on the quadratic polynomial equations using Origin 8.5 software. The response surface plots of DL (a) and EE (b), the response surface contour plots of DL (c) and EE (d), the overlay chart (e) between (c) and (d).
Both equations can describe all the dependent variables exactly with high correlation coefficients (R40.95, p50.05). Then response surface plots (Figure 1a and b) and corresponding contour maps (Figure 1c and d) were depicted based on quadratic polynomial equations using Origin 8.5 software (Northampton, USA). It could be received from the plots that both DL% and EE% had the optimal areas, and the best chosen area would be obtained through the overlap of the two contour maps just shown in Figure 1(e). Considering higher DL and EE, several experiments were
carried out on the basis of the optimal area to find out the ideal amount of CUR and the mass ratio of PC to SDC. The final optimum results were as follows: X1 ¼ 17.9 mg, X2 ¼ 0.264. According to the optimum consequences, the solubility of CUR was 3.14 mg/ml, which meant that CUR was solubilized significantly compared with bulk drug in water. To test and verify the established mathematic model, the data received from experiments were compared with those from model (shown in Table 2). Each experiment was
Curcumin-loaded mixed micelles
DOI: 10.3109/10717544.2013.873501
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Table 2. Observed results and predicted results of DL and EE based on the optimal preparation technology of CUR-PC-SDC-MMs. Dependent variable
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DL (%) EE (%)
Predicted
Observed
Bias (%)
13.53 87.60
13.48 87.13
0.37 0.54
Figure 3. Plot of the fluorescence intensity ratio between the emission wavelength 372 nm and 383 nm versus the logarithm of concentration of mixed micelles in the PBS (pH 7.4) at room temperature.
Figure 2. The morphology images of CUR-PC-SDC-MMs under TEM.
performed in triplicate. The smaller relative deviation illustrated the prediction of mathematic model was good, and the results of the experimental design were of high reliability. Morphology, particle size and zeta potential of mixed micelles Under TEM, mixed micelles presented spherical in shape with no conglutination and were distributed uniformly (shown in Figure 2). The mean particle size of the optimal mixed micelles was 66.5 ± 1.5 nm with a polydispersity index of 0.216 ± 0.008. The average zeta potential measured was 26.96 ± 0.95 mV.
Figure 4. Profiles of the cumulative release percentage of curcumin from mixed micelles (g) and the propylene glycol solution () at pre-set time point in physiological saline containing 1% Tween 80.
Critical micelle concentration Both PC and sodium cholate were amphiphilic molecules with the capability of self assembly in aqueous solution just above the CMC. Pyrene fluorescence probe method was used to measure the CMC of mixed micelles with the mass ratio of PC to SDC of 0.264. With the logarithm of concentration of mixed micelles as the abscissa and the ratio of I372 to I383 as the ordinate, the CMC was 0.0087 g/l gained from intersection of the two tangents to scatter diagram (shown in Figure 3). In vitro release Figure 4 exhibited the dissolution profile of CUR from mixed micelles and propylene glycol solution in physiological saline containing 1% Tween 80 (w/v) with 12.3% of incorporated CUR was released within the first two hours in experimental group, which showed the burst release because of the drug absorbed on the surface of micelles. Ninety-six percent of CUR in propylene glycol solution escaped rapidly from
dialysis bag within 12 h. However, only 46.3% was released from the micellar system during the same time. After 72 hours, 24.6% of the initially incorporated drug was still detained in mixed micelles, indicating that CUR stayed tightly and stably inner core. Cell toxicity assays The cell toxicity of CUR-PC-SDC-MMs was determined by the MTT assay with experimental group (CUR-PC-SDCMMs), control group (free CUR dissolved in DMSO) and empty control group (mixed micelles without drug) all at the same concentration gradient. Because MCF-7 cell lines were sensitive and inhibited by CUR (Banerjee et al., 2010), they were selected to perform the toxicity experiment. Cell inhibition percentage at different concentrations of three kinds of agents was shown in Figure 5. From the plot, it can be concluded that the cell growth inhibition between CUR inside mixed micelles and free CUR had no significant
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Figure 6. The schematic illustration of the spherical structure of CUR-PC-SDC-MMs. Figure 5. Plot of the cell inhibition percentage of MCF-7 cells exposed to drug-loaded micelles (), empty micells (m) and free drug (g) at different concentration for 48 h.
differences at the concentration range from 0.75 to 3 mg/ml and 12 to 24 mg/ml; however, great changes occurred from 3 to 12 mg/ml. Empty micelles, just containing PC and bile salt, also represented a certain cell toxicity probably owing to the toxicity of SDC to epithelia and membranes, which could be counteracted partly by PCs (Dial et al., 2008). The IC50 values on MCF-7 cells of CUR-PC-SDC-MMs and free drug solution were 4.10 mg/ml and 6.93 mg/ml, respectively.
Discussion CUR-loaded bile salt/PC mixed micelles have been proved a successful injection preparation to improve the solubility in the aqueous solution. High DL% and EE% maybe ascribed to the potential mechanization that PC and SDC reveal the strongest bonding force (Duan et al., 2011) when the mass ratio is at 0.264. The aggregation system of PCs alone is liquid crystalline bilayer structure instead of micelle, which is difficult to be digested (Tang et al., 2009). Nevertheless, Walter et al. (1991) reported that bile salts had an effect of solubilization on PCs to form mixed micelles, the hydrophobic region of bile salts interacted with the PC acyl chain region to form the hydrophobic core and their polar area formed the hydrophilic shell in contact with water. This effect was dependent on the amount of bile salts to a certain extent (Mazer et al., 1980), the DL% efficiency and drug solubility were enhanced significantly with the decrease of the mass ratio of PC and SDC. Although free bile salts could also form micelles to solubilize the insoluble drugs, the solubilized ability was far lower than that of the mixed micelles. Thus, the optimized mass ratio of PC to SDC got from the experiment basically accorded with the above discussion. The micro-morphology showed that the CURPC-SDC-MMs prepared through thin film dispersed method were spherical, which is consistent with the previous report (Duan et al., 2011). In terms of the aggregate structure, lots of researchers have studied on bile salt/PC mixed micelles by a series of methods such as static and dynamic light scattering, small-angle neutron scattering, small angle x-ray scattering and cryo-TEM (Long et al., 1994). The main structure theory
was ‘‘mixed disk model’’ with PC bilayer in the center and bile salts surrounding the perimeter of micelles (Attwood & Florence, 1983). Hjelm et al. (1988) put forward a new theory about cylindrical structure of bile salt/PC micelles. Along the aggregate axis, PCs were distributed radically with bile salts laying flat at the interface (Hjelm et al., 1992). However, the CUR-PC-SDC-MMs were spherical uniformly under TEM. Based on the above two theories, the CUR-loaded mixed micelles were inferred spherical structures that PC molecules formed monolayer perforated with SDCs surrounding the spherical surface. When faced with the acyl chains of PCs, SDCs had two oxhydryl groups in hydrophobic region which led to the unfavorable situation (Zhou et al., 2010). Therefore, bile salts tended to gather to form a dimer to protect the oxhydryl groups from high hydrophobic area (Wu & Wang, 2011). As shown in Figure 6, the hydrophobic cores of micelles consisted of the acyl chains of PC and the hydrophobic region of SDC dimers, whose hydrophilic regions formed the shells. In addition, CUR was packaged in the core through physical coating, hydrogen bonds or van der Waals force. The size of nanoparticles and zeta potential on the colloid surfaces were evaluated later. Particle size could influence tissue distribution of colloids in vivo (Sezgin et al., 2006). Usually, kidneys in healthy body have the ability to filter the smaller particles with size less than 10 nm, and particles larger than 100 nm would be captured by healthy liver (Yu et al., 2010). Thus, the ideal particle size range is from 10 to 100 nm, which may lengthen the circulation time of particle in blood. At the same time, the appropriate size can enhance the accumulation of micelles in tumor tissue for enhanced permeability and retention effect. Therefore, the size of CURPC-SDC-MMs was suitable for the passive tumor-targeting transport to play an antitumor effect, and the mixed micelles could be safely separated from the excretory and detoxifying organs. Similar to particle size, the zeta potential was also of importance to the colloid system. It was a significant index to characterize the stability of colloidal dispersion (Komatsu et al., 1995). Commonly, zeta potential values of 30 mV (absolute value) or above would provide a stable environment since the electrical repulsion forces could inhibit the aggregation of micelles (Mu¨ller et al., 2000; Liu et al., 2007).
Curcumin-loaded mixed micelles
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DOI: 10.3109/10717544.2013.873501
From the result, the surface of mixed micelles was negatively charged, which perhaps revealed the anion surfactants (SDC) surrounded the micelles. Meanwhile, the absolute value of surface electric charge was close to 30 mV enough to repulse mutually between particles for keeping system stable (Zhao et al., 2012). Based on the principle that pyrene emission spectra was dependent on the polarity of the microenvironment, the hydrophobic pyrene was encapsulated into the core of micelle at different concentrations resulting in the decrease of the ratio between I372 and I383 (Me´nard et al., 2012). The CMC of CUR-loaded mixed micelles from the experiment was 0.0087 g/l, far lower than the CMC of SDC (3 mM) (Zhou et al., 2010). This phenomenon may be explained as the strong interaction forces between PC and SDC, as mentioned above, which promoted the ability to form micelles spontaneously, which indicated mixed micelles had better solubilization, stability and ability of anti-dilution (Chen et al., 2001). To simulate the release process in vivo, the release characteristic in vitro of CUR-loaded mixed micelles was carried on in physiological saline added with 1% Tween 80 in order to enhance the solubility of CUR. The release of CUR from micellar system represented sustained release characteristic compared to the relative rapid release in the control group. The interaction forces between CUR and carriers made CUR stable in the simulated physiological environment to some extent. On the other hand, the novel drug delivery system would become a favorable injection preparation. The cytotoxicity of CUR-PC-SDC-MMs was obvious on MCF-7 cells with an IC50 value of 4.10 mg/ml. Based on the above results, not only drug but also excipients acted on the MCF-7 cell. The inhibition effects on MCF-7 cell of CUR entrapped in micelles and free CUR below the concentration of 3 mg/ml were not obvious, and when the concentration of CUR was above 12 mg/ml, just free drug was enough to inhibit 80% or more cell growth, thus the interaction between carrier materials and cell membranes were not represented. Between 12 and 24 mg/ml, excipients, especially SDC, had great influences on the cell inhibition. The increasing of cell plasma membrane permeability induced by excipients (Zhou et al., 2010) led to the more permeation of drug into the cell, which could be explained for the different cell inhibition between drug-loaded micelles and free drug. On the other hand, the better stability and solubility of CUR inside mixed micelles, compared with bulk drug, also contributed to the differences above. In summary, the micellar system could enhance the anti-tumor ability of CUR. Moreover, the toxicity of empty micelles to cell membrane still ought to attract attention based on the consequences from the experiment. In consideration of the significant solubilization of SDC toward mixed micelles, a low mass ratio of PC to SDC was put to use in this experiment. On the other hand, the amount of SDC needs appropriate adjustment when used in practice.
Conclusion In this study, CUR-loaded PC–SDC-mixed micelles were prepared through the thin-film dispersion method. As a result of the formulation optimization, the micelles exhibited spherical shape, smaller size and negatively charged colloid
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surface. In addition, the CMC of carrier materials, including PC and SDC, was so low that the integrality of micelles was ensured when they were in touch with body fluids after injection administration. The release experiment in vitro displayed a sustained release property of micelles, which enhanced the circulation time in blood. The cytotoxicity assays of CUR-loaded micelles on MCF-7 cells showed the higher cell toxicity compared with free CUR. In conclusion, the PC–SDC micelles developed in this study were proved a promising drug delivery system for CUR.
Declaration of interest The authors report no conflicts of interest. This work is supported by the Natural Science Foundation of Shandong Province, China (No.ZR2011HM026) and the National Natural Science Foundation of China (No.30973646).
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Kuttan R, Bhanumathy P, Nirmala K, George MC. (1985). Potential anticancer activity of turmeric (Curcuma longa). Cancer Lett 29: 197–202. Liu J, Gong T, Wang CG, et al. (2007). Solid lipid nanoparticles loaded with insulin by sodium cholate-phosphatidylcholine-based mixed micelles: preparation and characterization. Int J Pharm 340:153–62. Long MA, Kaler EW, Lee SP. (1994). Structural characterization of the micelle-vesicle transition in lecithin-bile salt solutions. Biophys J 67: 1733–42. Mahady GB, Pendland SL, Yun G, Lu ZZ. (2001). Turmeric (Curcuma longa) and curcumin inhibit the growth of Helicobacter pylori, a group 1 carcinogen. Anticancer Res 22:4179–81. Mattila MAK, Suistomaa M. (1984). Intravenous premedication with diazepam: a comparison between two vehicles. Anaesthesia 39:879–82. Mazer NA, Benedek GB, Carey MC. (1980). Quasielastic lightscattering studies of aqueous biliary lipid systems. Mixed micelle formation in bile salt-lecithin solutions. Biochemistry 19:601–15. Me´nard N, Tsapis N, Poirier C, et al. (2012). Drug solubilization and in vitro toxicity evaluation of lipoamino acid surfactants. Int J Pharm 423:312–20. Mu¨ller RH, Ma¨der K, Gohla S. (2000). Solid lipid nanoparticles (SLN) for controlled drug delivery – a review of the state of the art. Eur J Pharm Biopharm 50:161–77. Sezgin Z, Yuksel N, Baykara T. (2006). Preparation and characterization of polymeric micelles for solubilization of poorly soluble anticancer drugs. Eur J Pharm Biopharm 64:261–8. Sharma OP. (1976). Antioxidant activity of curcumin and related compounds. Biochem Pharmacol 25:1811–12. Shishodia S, Sethi G, Aggarwal BB. (2005). Curcumin: getting back to the roots. Ann N Y Acad Sci 1056:206–17. Srimal RC, Dhawan BN. (1973). Pharmacology of diferuloyl methane (curcumin), a non-steroidal anti-inflammatory agent*. J Pharm Pharmacol 25:447–52.
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Su DS, Wang SL. (2004). Physical pharmacy. Beijing, CN: Beijing Chemical Industry Press. Sugioka H, Moroi Y. (1999). Micelle formation of sodium cholate and solubilization into the micelle. Biochim Biophys Acta 1394: 99–110. Tang N, Lai J, Chen YP, et al. (2009). Fenofibrate solid dispersion pellets prepared by fluid-bed coating: physical characterization, improved dissolution and oral bioavailability in beagle dogs. J Chin Pharm Sci 18:156–61. Walter A, Vinson PK, Kaplun A, Talmon Y. (1991). Intermediate structures in the cholate-phosphatidylcholine vesiclemicelle transition. Biophys J 60:1315–25. Wiedmann TS, Kamel L. (2002). Examination of the solubilization of drugs by bile salt micelles. J Pharm Sci 91:1743–64. Wiedmann TS, Liang W, Kamel L. (2002). Solubilization of drugs by physiological mixtures of bile salts. Pharm Res 19:1203–8. Wu TH, Wang ZN. (2011). Micellization and phase behavior of biosurfactant bile salts. Prog Chem 23:80–9. Yu JN, Zhu Y, Wang L, et al. (2010). Enhancement of oral bioavailability of the poorly water-soluble drug silybin by sodium cholate/ phospholipid-mixed micelles. Acta Pharmacologica Sinica 31: 759–64. Zhao LY, Du JC, Duan YW, et al. (2012). Curcumin loaded mixed micelles composed of pluronic P123 and F68: preparation, optimization and in vitro characterization. Coll Surf B 97:101–8. Zhou Y, Dial EJ, Doyen R, Lichtenberger LM. (2010). Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs. Am J Physiol Gastrointest Liver Physiol 298:G722–31. Zhu Y, Yu JN, Tong SS, et al. (2010). Preparation and in vitro evaluation of povidone-sodium cholate-phospholipid mixed micelles for the solubilization of poorly soluble drugs. Arch Pharm Res 33:911–17.
ORIGINAL ARTICLE
Curcumin-loaded mixed micelles: preparation, optimization, physicochemical properties and cytotoxicity in vitro Yuwei Duan1, Juan Wang2, Xiaoye Yang1, Hongliang Du1, Yanwei Xi1, and Guangxi Zhai1 Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan, China and 2Department of Pharmacy, Jinan Maternity and Child Care Hospital, Jinan, China
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Abstract
Keywords
Although curcumin (CUR) can inhibit proliferation and induce apoptosis of tumors, the poor water solubility restricted its clinical application. The aim of this study was to improve the aqueous solubility of CUR and make more favorable changes to bioactivity by preparing curcumin-loaded phospholipid-sodium deoxycholate-mixed micelles (CUR-PC-SDC-MMs). CUR-PC-SDC-MMs were prepared by the thin-film dispersion method. Based on the results of single factor exploration, the preparation technology was optimized using the central composite design-response surface methodology with drug loading and entrapment efficiency (EE%) as indicators. The images of transmission electron microscopy showed that the optimized CUR-PC-SDC-MMs were spherical and well dispersed. The average size of the mixed micelles was 66.5 nm, the zeta potential was about 26.96 mV and critical micelle concentration was 0.0087 g/l. CUR was encapsulated in PC-SDC-MMs with loading capacity of 13.12%, EE% of 87.58%, and the solubility of CUR in water was 3.14 mg/ml. The release results in vitro showed that the mixed micelles presented sustained release behavior compared to the propylene glycol solution of CUR. The IC50 values of CUR-loaded micelles and free drug in human breast carcinoma cell lines were 4.10 mg/ml and 6.93 mg/ml, respectively. It could be concluded from the above results that the CUR-PC-SDC-MMs system might serve as a promising nanocarrier to improve the solubility and bioactivity of CUR.
Curcumin, cytotoxicity, drug delivery system, phospholipid-sodium deoxycholate, solubility
Introduction The mixed micellar system formed by different surface active agents mixed with each other, because of the better performance than single surfactant, has attracted increasing attention in recent years (Zhu et al., 2010). Bile salts and phospholipids (PC) as anionic and amphoteric surfactants are both amphipathic molecules, which can show the characteristic of self assembly in the aqueous solution (Hernell et al., 1990; Sugioka & Moroi, 1999; Su & Wang, 2004). For organism, bile salts, secreted by liver and stored in the gall bladder, could form a kind of stable mixed micellar system with PCs, glycerides and fatty acids during lipid digestion in the small intestine (Yu et al., 2010; Duan et al., 2011). The bile salt/PC micelles can package cholesterol inside the hydrophobic core, and the solubility, stability and bioavailability of poorly water-soluble drugs could be enhanced in the same way (Dangi et al., 1995; Wiedmann & Kamel, 2002; Wiedmann et al., 2002; Garidel et al., 2007). Meanwhile, water-soluble drugs would insert into the hydrophilic palisade
Address for correspondence: Guangxi Zhai, PhD, Department of Pharmaceutics, College of Pharmacy, Shandong University, 44 Wenhua Xilu, Jinan 250012, China. Tel: (86) 531-88382015. Fax: (86) 531-88382731. Email: [email protected]
History Received 13 October 2013 Revised 29 November 2013 Accepted 5 December 2013
layer of micelles to separate from the surrounding unavailable environment. Many investigators have successfully improved the solubility and bioavailability of insoluble drugs in this way, such as fat-soluble vitamins, paclitaxel, lorazepam and diazepam (Chen et al., 2001). Besides the advantages above, bile salts, PCs and their degradation products, as components existing in organism, could be absorbed by human body completely. As a result, the combination of bile salts and PCs, which raises the safety of preparation, is considered as biocompatible injection solvent (Mattila & Suistomaa, 1984) and a promising delivery system for anticancer drugs. Curcumin (CUR), a yellow acidic polyphenol compound, is an effective active ingredient extracted from the rhizome of the perennial herb Curcuma longa (Gandapu et al., 2011). It has been used in traditional medicine for many years with a wide range of pharmacological actions (Shishodia et al., 2005), such as anti-inflammatory, antioxidant, anti-microbial and anti-tumoral effects (Srimal & Dhawan, 1973; Sharma, 1976; Kuttan et al., 1985; Mahady et al., 2001). CUR is capable to defend against cancer in vitro and in vivo through multiple mechanisms. It can prevent the proliferation and induce apoptosis of various tumor cells (Aggarwal et al., 2003). High doses of CUR (12 g/d) given to clinical models are better tolerated, which shows CUR is safe for organism (Hsu & Cheng, 2007). However, whether oral administration or injection, the bioavailability of CUR is poor with
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properties of insoluble in water, easily metabolized and fast systemic eliminated, which has become the main block of the clinical application (Anand et al., 2007). Improving the solubility, stability and bioavailability is of significant importance to the wide usage of CUR as therapeutic agent. Until now, a series of dosage forms for CUR have been developed including nanosuspension, nanoparticles, nanospheres and inclusion complex, etc. Based on the shortcomings of CUR and the superiority of bile salt/PC-mixed micelles, this article reports a new injectionable micelle formulation to enhance the solubility and bioactivity of CUR. Curcumin-loaded phospholipid-sodium deoxycholate mixed micelles (CURSDC-PC-MMs) were prepared by the thin-film dispersion method. Then the preparation was optimized with the central composite design (CCD)-response surface method. In addition, the physicochemical properties and the cell toxicity of CUR-PC-SDC-MMs in vitro were investigated.
Materials and methods Materials CUR and SDC were purchased from Sigma-Aldrich (St. Louis, MO). Soybean lecithin (PC) was purchased from Shang Hai Tai Wei Pharmaceutical Co., Ltd. (Shanghai, China). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), trypsin and ethylenediaminetetraacetic acid were purchased from Amresco (Solon, OH). RPMI 1640 was purchased from Thermo Fisher (Beijing, China). Fetal bovine serum (FBS) was purchased from Tian Jin Hao Yang Biological Manufacture Co., Ltd. (Tianjin, China). Human breast carcinoma cell lines (MCF-7) were donated by Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University. All other chemicals were of analytical grade.
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Study on physicochemical properties Particle size and zeta potential Particle size and zeta potential of CUR-PC-SDC-MMs were measured using DelsaNano-based-Beckman coulter at 25 C. Each sample was measured in triplicate. Micro-morphology The morphology of optimum CUR-PC-SDC-MMs was observed by the transmission electron microscopy (TEM). A drop of the formulation was placed on a copper grid and stained with phosphotungstic acid solution (2%, w/v) for 15 s. Then, the excess solution was absorbed, and the sample was dried in air and examined under TEM. Drug loading and EE% The content of CUR encapsulated in PC-SDC-MMs was determined by UV-Vis spectrophotometer at 426 nm. The prepared micellar solution was diluted to a certain concentration with anhydrous ethanol before determination. The drug loading (DL%) and entrapment efficiency (EE%) were calculated by the following equations (Jansson et al., 2004): WCUR DL% ¼ 100% ð1Þ Wmicelle WCUR 100% ð2Þ WCUR0 where WCUR, the weight of drug in micelles; WCUR’, the weight of feeding drug; and Wmicelle, the total weight of feeding PC, SDC and drug in micelles. It is reported that the solubility of CUR in water was 11 ng/ml (Cui et al., 2009), much smaller compared with loading content of CUR inside micelles, so DL% and EE% were calculated ignoring the content of CUR in water (Chen et al., 2001). EE% ¼
Preparation of CUR-PC-SDC-MMs
Critical micelle concentration
CUR-PC-SDC-MMs were formed through thin film dispersed method. Briefly, CUR, PC and SDC (with different concentrations and proportions) were dissolved in anhydrous ethanol by stirring at room temperature. Then, the solution was evaporated at 40 C under reduced pressure to obtain a thin film of drug-carrier materials, and the film was further dried in vacuum drying oven over night to remove redundant organic solvent. After that, the film was hydrated in 5 ml PBS and dispersed for 15 min via ultrasound. The micellar suspension was centrifuged at 12 000rpm for 5 min in order to separate unloaded drugs, and the supernatant was collected, which contained CUR-PC-SDC-MMs. The empty micelles were prepared in the same way just without CUR.
The critical micelle concentration (CMC) of PC-SDC in the PBS (pH 7.4) was determined with pyrene fluorescence probe method using F-2500 fluorescence spectrophotometer. The prepared blank micelles were diluted to a series of concentrations ranging from 1 103 to 2 g/l. Pyrene dissolved in acetone was added to a few of amber glass vials, and the acetone was evaporated with continuous flow of nitrogen gas (Chen et al., 2001). Micellar solutions with different concentrations were added into the amber glass vials with a final pyrene concentration of 2 107 mol/l, and the solution was mixed uniformly. Then the mixtures were incubated for balance in the dark for 24 h at room temperature prior to measurement. Fluorescence emission spectrum was scanned from 350 to 450 nm with the rate at 1200 nm/min, at an excitation wavelength of 334 nm. The slits of excitation and emission were 5 nm and 2.5 nm, respectively. The fluorescence intensity at wavelength 372 and 383 nm was recorded, respectively, and the ratio between I372 and I383 would be alternated with pyrene moving into the interior of micelles. All experiments were performed in triplicate.
Optimization of the preparation technology In order to optimize the preparation technology, the CCDresponse surface method was used based on single-factor experiments such as the amount of CUR added, the mass ratio of PC to SDC, the volume of hydration solution, hydration temperature and time.
Curcumin-loaded mixed micelles
DOI: 10.3109/10717544.2013.873501
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In vitro release of CUR The release behavior of CUR from micellar system was studied with physiological saline containing 1% Tween 80 (w/v) as release medium using dialysis method. Under the premise of sink condition, an appropriate volume of CUR-PC-SDC-MMs and CUR solution (dissolved in propylene glycol) as control, with an equal content of CUR, was placed into a pre-swollen dialysis membrane bag. The dialysis bag was tied and placed into an Erlenmeyer flask containing 50 ml of release medium. The temperature and stirring speed were set at 37 C and 120 r/min separately. At each pre-set time point, 1 ml of the medium was withdrawn from the flask and 1 ml of fresh medium was added into the flask. The samples taken out were filtered through 0.22 mm filter membrane, and the filter liquor was measured by high-performance liquid chromatography (HPLC) at 426 nm. A Hypersil BDS C18 reserved phase analytical column (5 mm, 4.6 mm 250 mm) was applied in HPLC analyses. The mobile phase consisted of methanol/ water containing 3.6% glacial acetic acid (70:30) was pumped at the rate of 1.0 ml/min. The column temperature was set at 25 C. The cumulative release percentage of CUR was calculated. Every release experiment was repeated three times. Cell toxicity assays MCF-7 cells were adopted to evaluate the in vitro toxicity of CUR-PC-SDC-MMs with the MTT method. Cells were cultured in the RPMI-1640 medium supplemented with 10% FBS. All the cells were grown at 37 C in 5% CO2 atmosphere in a humidified incubator, and subcultured once every two days. MCF-7 cell lines were seeded in 96-well culture plates at the density of 5 103 per well. After 24 h of incubation, cells were handled with CUR-PC-SDC-MMs of different concentrations, blank micelles and CUR solution dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was below 0.2% according to references (Zhao et al., 2012), and the concentrations of treatment agents ranged from 0.75 mg/ml to 24 mg/ml. After incubation for 48 h, medium in well was withdrawn and phosphate buffer saline was added to wash the well. Next, each well was
replenished with fresh culture medium with 20 ml of MTT (5 mg/ml) in it. The culture plates were incubated for 4 h at 37 C and centrifuged for 10 min at 3000 rpm/min. Subsequently, the medium was moved out and 150 ml of DMSO was added to dissolve the formazan crystals produced inside cells. Absorbance of cells were measured by multiwell scanning spectrophotometer Model 680 (Bio-Rad, Hercules, CA) at 570 nm and 630 nm (Zhao et al., 2012). Each concentration was set up in six replicates, and experiment was measured in triplicate. The formula of cell inhibition percentage was as follows: Absorbanceexperimental Cell Inhibition % ¼ 1 100% Absorbancecontrol where Absorbanceexperimental, the absorbance of cells interacted with micelles or drug; Absorbancecontrol, the absorbance of cells cultured with no micelles or drug.
Results Optimization of the preparation technology Taking DL% and EE% as index, a series of single-factor experiments were performed to select the control factors, which influenced preparation technology significantly. The results indicated that DL% and EE% had obvious changes along with varying amount of CUR and the mass ratio of PC to SDC. Hence, the two factors were conducted as variables for optimization through CCD at five experimental levels. Amount of CUR was as X1 ranging from 1.25 to 20 mg, and the mass ratio of PC to SDC was as X2 in the range from 0 to 2.333. The experimental design and results were shown in Table 1. The data was dealt with SPSS 17.0 (SPSS Inc., Chicago, IL) statistical software making multivariate nonlinear regression. The quadratic polynomial equations were obtained as follows: YDL% ¼ 1:127 þ 1:018X1 þ 2:305X2 0:011X21 0:790X22 0:126X1 X2 YEE% ¼ 86:879 þ 19:200X2 0:799X1 X2 8:243X22
Table 1. Central composite design with code values and observed values. Code values of variables
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Actual values of variables
Observed
No.
X1
X2
Amount of CUR
the mass ratio of PC and SDC
DL (%)
EE (%)
1 2 3 4 5 6 7 8 9 10 11 12 13
1 1 1 1 1.414 1.414 0 0 0 0 0 0 0
1 1 1 1 0 0 1.414 1.414 0 0 0 0 0
17.255 17.255 3.995 3.995 20.000 1.250 10.625 10.625 10.625 10.625 10.625 10.625 10.625
1.991 0.342 1.991 0.342 1.167 1.167 2.333 0 1.167 1.167 1.167 1.167 1.167
10.38 13.10 3.51 3.47 13.87 1.26 6.31 8.52 8.55 8.53 8.50 8.53 8.65
66.53 87.19 91.01 92.15 81.35 94.48 63.10 87.44 87.41 87.03 86.84 87.22 89.38
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Figure 1. Response surface plots and response surface contour plots based on the quadratic polynomial equations using Origin 8.5 software. The response surface plots of DL (a) and EE (b), the response surface contour plots of DL (c) and EE (d), the overlay chart (e) between (c) and (d).
Both equations can describe all the dependent variables exactly with high correlation coefficients (R40.95, p50.05). Then response surface plots (Figure 1a and b) and corresponding contour maps (Figure 1c and d) were depicted based on quadratic polynomial equations using Origin 8.5 software (Northampton, USA). It could be received from the plots that both DL% and EE% had the optimal areas, and the best chosen area would be obtained through the overlap of the two contour maps just shown in Figure 1(e). Considering higher DL and EE, several experiments were
carried out on the basis of the optimal area to find out the ideal amount of CUR and the mass ratio of PC to SDC. The final optimum results were as follows: X1 ¼ 17.9 mg, X2 ¼ 0.264. According to the optimum consequences, the solubility of CUR was 3.14 mg/ml, which meant that CUR was solubilized significantly compared with bulk drug in water. To test and verify the established mathematic model, the data received from experiments were compared with those from model (shown in Table 2). Each experiment was
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DOI: 10.3109/10717544.2013.873501
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Table 2. Observed results and predicted results of DL and EE based on the optimal preparation technology of CUR-PC-SDC-MMs. Dependent variable
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DL (%) EE (%)
Predicted
Observed
Bias (%)
13.53 87.60
13.48 87.13
0.37 0.54
Figure 3. Plot of the fluorescence intensity ratio between the emission wavelength 372 nm and 383 nm versus the logarithm of concentration of mixed micelles in the PBS (pH 7.4) at room temperature.
Figure 2. The morphology images of CUR-PC-SDC-MMs under TEM.
performed in triplicate. The smaller relative deviation illustrated the prediction of mathematic model was good, and the results of the experimental design were of high reliability. Morphology, particle size and zeta potential of mixed micelles Under TEM, mixed micelles presented spherical in shape with no conglutination and were distributed uniformly (shown in Figure 2). The mean particle size of the optimal mixed micelles was 66.5 ± 1.5 nm with a polydispersity index of 0.216 ± 0.008. The average zeta potential measured was 26.96 ± 0.95 mV.
Figure 4. Profiles of the cumulative release percentage of curcumin from mixed micelles (g) and the propylene glycol solution () at pre-set time point in physiological saline containing 1% Tween 80.
Critical micelle concentration Both PC and sodium cholate were amphiphilic molecules with the capability of self assembly in aqueous solution just above the CMC. Pyrene fluorescence probe method was used to measure the CMC of mixed micelles with the mass ratio of PC to SDC of 0.264. With the logarithm of concentration of mixed micelles as the abscissa and the ratio of I372 to I383 as the ordinate, the CMC was 0.0087 g/l gained from intersection of the two tangents to scatter diagram (shown in Figure 3). In vitro release Figure 4 exhibited the dissolution profile of CUR from mixed micelles and propylene glycol solution in physiological saline containing 1% Tween 80 (w/v) with 12.3% of incorporated CUR was released within the first two hours in experimental group, which showed the burst release because of the drug absorbed on the surface of micelles. Ninety-six percent of CUR in propylene glycol solution escaped rapidly from
dialysis bag within 12 h. However, only 46.3% was released from the micellar system during the same time. After 72 hours, 24.6% of the initially incorporated drug was still detained in mixed micelles, indicating that CUR stayed tightly and stably inner core. Cell toxicity assays The cell toxicity of CUR-PC-SDC-MMs was determined by the MTT assay with experimental group (CUR-PC-SDCMMs), control group (free CUR dissolved in DMSO) and empty control group (mixed micelles without drug) all at the same concentration gradient. Because MCF-7 cell lines were sensitive and inhibited by CUR (Banerjee et al., 2010), they were selected to perform the toxicity experiment. Cell inhibition percentage at different concentrations of three kinds of agents was shown in Figure 5. From the plot, it can be concluded that the cell growth inhibition between CUR inside mixed micelles and free CUR had no significant
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Figure 6. The schematic illustration of the spherical structure of CUR-PC-SDC-MMs. Figure 5. Plot of the cell inhibition percentage of MCF-7 cells exposed to drug-loaded micelles (), empty micells (m) and free drug (g) at different concentration for 48 h.
differences at the concentration range from 0.75 to 3 mg/ml and 12 to 24 mg/ml; however, great changes occurred from 3 to 12 mg/ml. Empty micelles, just containing PC and bile salt, also represented a certain cell toxicity probably owing to the toxicity of SDC to epithelia and membranes, which could be counteracted partly by PCs (Dial et al., 2008). The IC50 values on MCF-7 cells of CUR-PC-SDC-MMs and free drug solution were 4.10 mg/ml and 6.93 mg/ml, respectively.
Discussion CUR-loaded bile salt/PC mixed micelles have been proved a successful injection preparation to improve the solubility in the aqueous solution. High DL% and EE% maybe ascribed to the potential mechanization that PC and SDC reveal the strongest bonding force (Duan et al., 2011) when the mass ratio is at 0.264. The aggregation system of PCs alone is liquid crystalline bilayer structure instead of micelle, which is difficult to be digested (Tang et al., 2009). Nevertheless, Walter et al. (1991) reported that bile salts had an effect of solubilization on PCs to form mixed micelles, the hydrophobic region of bile salts interacted with the PC acyl chain region to form the hydrophobic core and their polar area formed the hydrophilic shell in contact with water. This effect was dependent on the amount of bile salts to a certain extent (Mazer et al., 1980), the DL% efficiency and drug solubility were enhanced significantly with the decrease of the mass ratio of PC and SDC. Although free bile salts could also form micelles to solubilize the insoluble drugs, the solubilized ability was far lower than that of the mixed micelles. Thus, the optimized mass ratio of PC to SDC got from the experiment basically accorded with the above discussion. The micro-morphology showed that the CURPC-SDC-MMs prepared through thin film dispersed method were spherical, which is consistent with the previous report (Duan et al., 2011). In terms of the aggregate structure, lots of researchers have studied on bile salt/PC mixed micelles by a series of methods such as static and dynamic light scattering, small-angle neutron scattering, small angle x-ray scattering and cryo-TEM (Long et al., 1994). The main structure theory
was ‘‘mixed disk model’’ with PC bilayer in the center and bile salts surrounding the perimeter of micelles (Attwood & Florence, 1983). Hjelm et al. (1988) put forward a new theory about cylindrical structure of bile salt/PC micelles. Along the aggregate axis, PCs were distributed radically with bile salts laying flat at the interface (Hjelm et al., 1992). However, the CUR-PC-SDC-MMs were spherical uniformly under TEM. Based on the above two theories, the CUR-loaded mixed micelles were inferred spherical structures that PC molecules formed monolayer perforated with SDCs surrounding the spherical surface. When faced with the acyl chains of PCs, SDCs had two oxhydryl groups in hydrophobic region which led to the unfavorable situation (Zhou et al., 2010). Therefore, bile salts tended to gather to form a dimer to protect the oxhydryl groups from high hydrophobic area (Wu & Wang, 2011). As shown in Figure 6, the hydrophobic cores of micelles consisted of the acyl chains of PC and the hydrophobic region of SDC dimers, whose hydrophilic regions formed the shells. In addition, CUR was packaged in the core through physical coating, hydrogen bonds or van der Waals force. The size of nanoparticles and zeta potential on the colloid surfaces were evaluated later. Particle size could influence tissue distribution of colloids in vivo (Sezgin et al., 2006). Usually, kidneys in healthy body have the ability to filter the smaller particles with size less than 10 nm, and particles larger than 100 nm would be captured by healthy liver (Yu et al., 2010). Thus, the ideal particle size range is from 10 to 100 nm, which may lengthen the circulation time of particle in blood. At the same time, the appropriate size can enhance the accumulation of micelles in tumor tissue for enhanced permeability and retention effect. Therefore, the size of CURPC-SDC-MMs was suitable for the passive tumor-targeting transport to play an antitumor effect, and the mixed micelles could be safely separated from the excretory and detoxifying organs. Similar to particle size, the zeta potential was also of importance to the colloid system. It was a significant index to characterize the stability of colloidal dispersion (Komatsu et al., 1995). Commonly, zeta potential values of 30 mV (absolute value) or above would provide a stable environment since the electrical repulsion forces could inhibit the aggregation of micelles (Mu¨ller et al., 2000; Liu et al., 2007).
Curcumin-loaded mixed micelles
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DOI: 10.3109/10717544.2013.873501
From the result, the surface of mixed micelles was negatively charged, which perhaps revealed the anion surfactants (SDC) surrounded the micelles. Meanwhile, the absolute value of surface electric charge was close to 30 mV enough to repulse mutually between particles for keeping system stable (Zhao et al., 2012). Based on the principle that pyrene emission spectra was dependent on the polarity of the microenvironment, the hydrophobic pyrene was encapsulated into the core of micelle at different concentrations resulting in the decrease of the ratio between I372 and I383 (Me´nard et al., 2012). The CMC of CUR-loaded mixed micelles from the experiment was 0.0087 g/l, far lower than the CMC of SDC (3 mM) (Zhou et al., 2010). This phenomenon may be explained as the strong interaction forces between PC and SDC, as mentioned above, which promoted the ability to form micelles spontaneously, which indicated mixed micelles had better solubilization, stability and ability of anti-dilution (Chen et al., 2001). To simulate the release process in vivo, the release characteristic in vitro of CUR-loaded mixed micelles was carried on in physiological saline added with 1% Tween 80 in order to enhance the solubility of CUR. The release of CUR from micellar system represented sustained release characteristic compared to the relative rapid release in the control group. The interaction forces between CUR and carriers made CUR stable in the simulated physiological environment to some extent. On the other hand, the novel drug delivery system would become a favorable injection preparation. The cytotoxicity of CUR-PC-SDC-MMs was obvious on MCF-7 cells with an IC50 value of 4.10 mg/ml. Based on the above results, not only drug but also excipients acted on the MCF-7 cell. The inhibition effects on MCF-7 cell of CUR entrapped in micelles and free CUR below the concentration of 3 mg/ml were not obvious, and when the concentration of CUR was above 12 mg/ml, just free drug was enough to inhibit 80% or more cell growth, thus the interaction between carrier materials and cell membranes were not represented. Between 12 and 24 mg/ml, excipients, especially SDC, had great influences on the cell inhibition. The increasing of cell plasma membrane permeability induced by excipients (Zhou et al., 2010) led to the more permeation of drug into the cell, which could be explained for the different cell inhibition between drug-loaded micelles and free drug. On the other hand, the better stability and solubility of CUR inside mixed micelles, compared with bulk drug, also contributed to the differences above. In summary, the micellar system could enhance the anti-tumor ability of CUR. Moreover, the toxicity of empty micelles to cell membrane still ought to attract attention based on the consequences from the experiment. In consideration of the significant solubilization of SDC toward mixed micelles, a low mass ratio of PC to SDC was put to use in this experiment. On the other hand, the amount of SDC needs appropriate adjustment when used in practice.
Conclusion In this study, CUR-loaded PC–SDC-mixed micelles were prepared through the thin-film dispersion method. As a result of the formulation optimization, the micelles exhibited spherical shape, smaller size and negatively charged colloid
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surface. In addition, the CMC of carrier materials, including PC and SDC, was so low that the integrality of micelles was ensured when they were in touch with body fluids after injection administration. The release experiment in vitro displayed a sustained release property of micelles, which enhanced the circulation time in blood. The cytotoxicity assays of CUR-loaded micelles on MCF-7 cells showed the higher cell toxicity compared with free CUR. In conclusion, the PC–SDC micelles developed in this study were proved a promising drug delivery system for CUR.
Declaration of interest The authors report no conflicts of interest. This work is supported by the Natural Science Foundation of Shandong Province, China (No.ZR2011HM026) and the National Natural Science Foundation of China (No.30973646).
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