J Huazhong Univ Sci Technol［Med Sci］ 37(3):371-378,2017 10.1007/s11596-017-1742-8 J DOI Huazhong Univ Sci Technol［Med Sci］ 37(3):2017

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Therapeutic Effects of Curcumin Nanoemulsions on Prostate Cancer* Yan-bin GUAN (关延彬)1, 2#, Shu-yao ZHOU (周树瑶)1, Yu-qiong ZHANG (张玉琼)1, Jia-le WANG (王佳乐)1, Yu-dong TIAN (田雨冬)3, Yong-yan JIA (贾永艳)1, 2, Yan-jun SUN (孙彦君)1, 2# 1 School of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou 450001, China 2 Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province, Zhengzhou 450001, China 3 Department of Urology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2017

Summary: The therapeutic potential of curcumin (Cur) is hampered by its poor aqueous solubility and low bioavailability. The aim of this study was to determine whether Cur nanoemulsions enhance the efficacy of Cur against prostate cancer cells and increase the oral absorption of Cur. Cur nanoemulsions were developed using the self-microemulsifying method and characterized by their morphology, droplet size and zeta potential. The results showed that the cytotoxicity and cell uptake were considerably increased with Cur nanoemulsions compared to free Cur. Cur nanoemulsions exhibited a significantly prolonged biological activity and demonstrated better therapeutic efficacy than free Cur, as assessed by apoptosis and cell cycle studies. In situ single-pass perfusion studies demonstrated higher effective permeability coefficient and absorption rate constant for Cur nanoemulsions than for free Cur. Our study suggested that Cur nanoemulsions can be used as an effective drug delivery system to enhance the anticancer effect and oral bioavailability of Cur. Key words: curcumin; nanoemulsion; prostate cancer; anticancer efficacy; oral absorption

Curcumin (Cur) is a polyphenolic molecule extracted from the rhizome of the plant Curcuma longa and is widely used in China and India as a traditional herb[1–3]. Studies revealed that Cur possesses a wide range of pharmacological activities including anti-oxidant, anti-inflammatory, anti-cancer, and anti-depressant properties[4, 5]. It exhibits chemopreventive activities against cancer by anti-proliferation and pro-apoptosis, prevention of cell motility and metastasis both in vitro and in vivo[6–8]. Various clinical trials have been underway to examine the efficacy of Cur as a therapeutic agent against cancer[9]. Phase Ⅰ clinical trials already demonstrated the safety of Cur even at a high dose of 12 g/day[10]. In spite of its therapeutic potential, the poor water solubility, chemical instability in the alkaline medium, rapid metabolism and poor absorption in gastrointestinal tract severely hinder its anti-cancer efficacy. Many preclinical and clinical studies in mice, rats and humans revealed a low bioavailability of Cur[11–13]. A maximum serum concentration of 0.36 mg/mL after an intravenous injection of 10 mg/kg was reached, whereas 500 mg/kg orally administered Cur gave a maximum plasma concentration of 0.06 mg/mL, indicating Yan-bin GUAN, E-mail: [email protected] # Corresponding authors, Yan-bin GUAN, E-mail: [email protected]; Yan-jun SUN, E-mail: [email protected] * This project was supported by grants from the Doctoral Program Foundation of Henan University of Traditional Chinese Medicine (No. BSJJ2011-04) and Specialized Research Fund of Henan University of Traditional Chinese Medicine (No. 2014KYYWF-QN12).

that the oral bioavailability of Cur was only 1 %[7, 14]. Recent studies showed that Cur encapsulated in polymeric nanoparticles could improve the therapeutic efficacy, compared with free Cur, due to the increased solubility in aqueous media[2, 15, 16]. Cur-liposomal was found to significantly induce the apoptosis of both human umbilical vein endothelial cells (HUVECs) and metastatic melanoma cells (B16F10)[17]. Although various nanoformulations have been used to improve the anticancer potential of Cur, there are few reports on nanocarriers to enhance the anticancer efficacy of Cur following oral administration[18]. An appropriate oral formulation of Cur has always been desirable. Among the various formulations, the self-microemulsifying drug delivery system (SMEDDS) is particularly suited for lipophilic drugs. SMEDDS has attracted significant attention due to its promising ability to form oil-in-water (o/w) nanoemulsions with gentle agitation following dilution in aqueous phases. In human, the digestive motility of the stomach and intestine provides the agitation required for self-emulsification in vivo[19–22]. The spontaneous formation of an emulsion upon drug release in the gastrointestinal tract advantageously makes the drug dissolved. Recent reports on the Cur nanoemulsions failed to give a definite conclusion about the size and stability of physicochemical parameters under different storage and experimental conditions[20, 23, 24], which will greatly affect the anti-tumor activity of Cur. In addition, the anti-prostate cancer activity and the mechanism of action of Cur SMEDDS have not been fully investigated . In our previous study, we had developed Cur nanoemulsions for oral bioavailability enhancement.

372 Exhaustive optimization was carried out to develop a formulation comprising medium chain triglyceride (MCT), cremophor RH 40 (RH 40) and glycerol. The formulation showed better stability than free Cur and it could increase the solubility of Cur by 140 times[25]. In this study, we further investigated the oral absorption and anti-cancer potential of previously developed Cur nanoemulsions. 1 MATERIAL AND METHODS 1.1 Animals and Samples Pure Cur (99.9%) and methyl thiazolyl tetrazolium (MTT) were purchased from Sigma Aldrich (Switzerland). Cremophor RH40 was obtained from BASF (Germany). MCT, and glycerol as oil were purchased from Sinopharm Company in China, absolute ethanol (analytical grade) and methanol (chromatographic grade) from Shanghai Chemical Reagents Company, China. The human prostatic carcinoma cell line, PC-3, was procured from the Institute of Biochemistry and Cell Biology (IBCB), Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. RPMI 1640 medium, penicillin, streptomycin and amphothericin B were bought from Invitrogen Corp (USA), fetal calf serum (FCS) from ICN Biomedicals, Inc. (Osaka, Japan), and the annexin V-FITC apoptosis detection kit from Nanjing Keygen Biotech. Co. LTD., China. 1.2 Preparation and Characterization of Cur Nanoemulsions Cur nanoemulsions were prepared by using MCT, cremophor RH40 and glycerol as oil, surfactant and co-surfactant, respectively. Self-microemulsifying technology was used. Briefly, 10 mg of Cur was added to a glass vial containing the mixture (1 g) of MCT, cremophor RH40 and glycerol (22.5%:49.32%:28.18%), which was followed by vortexing for 2 min to obtain a homogenous mixture. The resultant mixture was allowed to be agitated with a magnetic stirrer at 37°C to form the Cur-SMEDDS. The mixture was stored at room temperature until used. Before using it for in-vitro test, the formulation was diluted with distilled water to form Cur nanoemulsions. 1.2.1 Particle Size and Zeta Potential Determination The particle size, polydispersity index (PDI) and zeta potential of Cur nanoemulsion were determined by photon correlation spectroscopy, using a Zetasizer 1000 HS (Malvern Instruments, UK). The samples (0.1 mL) were diluted with 5 mL of distilled water, mixed thoroughly and light scattering was carried out at an angle of 90° at 25°C. 1.2.2 Transmission Electron Microscopy (TEM) A drop of Cur nanoemulsions was placed on the surface of a copper grid, and the excess liquid was drained onto the filter paper. TEM graphs were obtained using a transmission electronic microscope (JEM 1400, JEOL, Japan) operated at 80 kV. 1.3 Cell Culture PC-3 cells were cultured in RPMI 1640 containing 10% FCS, 100 IU/mL penicillin, 0.1 mg/mL streptomycin and 25 µg/mL amphothericin B in a humidified atmosphere of 5% CO2 with 95% relative humidity at 37°C. The cells were grown in tissue culture flasks and the medium was changed every two

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days. At 80–90% confluence, they were treated with 0.25% trypsin. 1.4 Cell Cytotoxicity Assay For cytotoxicity testing, the cells were seeded in 96-well microtiter plates with 5000 cells/well and cultured over 24 h to allow reattachment. Different concentrations (10–100 µmol/L) of free Cur (in DMSO) or Cur nanoemulsions diluted in the medium were added to treat PC-3 cells for various time. The cells were then washed with PBS. 20 µL of MTT (10 mg/mL) was added to each well and the cells were incubated for 4 h at 37°C in the dark. 200 µL of DMSO solution was then added to each well to dissolve the MTT formazan crystals. Absorbance of each well was measured at 570 nm using a microplate reader (SpectraMax M5, Molecular Devices, USA) where wells without cells served as controls. The net absorbance was used as an index of cell viability. 1.5 Cell Uptake Studies Both qualitative and quantitative determinations of Cur uptake by PC-3 cells treated with free Cur or Cur nanoemulsions were done using fluorescence microscopy and high-performance liquid chromatography (HPLC), respectively. Briefly, PC-3 cells were seeded at a density of 2×105 cells per well in a 6-well plate for 24 h and were treated with 60 µmol/L Cur/well for different time intervals (0.5, 1, 3, 6, 9, 12, 24 h). At each time point, the solution was removed to terminate the uptake and the cells were collected by trypsinization and centrifuged at 3000 r/min for 3 min. The supernatant was decanted and the pellets were resuspended in 1 mL methanol and vortexed for 5 min to extract the Cur in methanol fraction. Then, the lysate was centrifuged at 12000 r/min for 5min and a 20 µL aliquot of the supernatant containing methanolic Cur was used for HPLC analysis. 1.6 HPLC Analysis of Cur An Agilent 1260 HPLC system (Agilent instruments, US) was used to measure Cur concentration. Separation was performed on a C18 column (4.6 mm × 250 mm; Agilent, USA) at 32°C with a flow rate of 0.9 mL/min. A UV-VIS Spectrophotometer was set at 428 nm. The mobile phase consisted of 0.5% glacial acetic acid in distilled water and methanol (6:4, v/v). The limit of detection (LOD) of Cur was 5 ng/mL. The calibration curves were linear (R2=0.9998) over the study concentrations of 0.1 to 5 µg/mL. 1.7 Cell Cycle Analysis by Flow Cytometry PC-3 cells were treated with the media containing free Cur or Cur nanoemulsions at various concentrations (10–100 µmol/L) for 24, 48 and 7 h. Other cells were left untreated as negative control. Both floating and attached cells were collected, washed in ice-cold PBS, and fixed with 70% ice-cold ethanol overnight at 4°C. Then, the cells were treated with RNase A (200 µg/mL in PBS) for 30 min at 37°C. Subsequently, cells were stained with 25 µg/mL PI at room temperature for 30 min in the dark. The untreated cells cultured in parallel were used as controls. The DNA content of the cells was determined by flow cytometry, and cell cycle distribution was analyzed using ModiFit and CellQuest software. 1.8 Apoptosis Analysis by Caspase 3/7 Activity The apoptosis of PC-3 cells induced by free Cur

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or Cur nanoemulsions was analyzed by using the Cell Meter Caspase 3/7 Activity Apoptosis Assay Kit (AAT Bioquest, Biomol, Germany). PC-3 cells were plated in 96-well microplates at a density of 20 000 cells per well and cultured over 24 h to allow reattachment. Then the cells were treated with the media containing free Cur or Cur nanoemulsions at 60 µmol/L for 24, 48 and 72 h. Untreated cells were used as negative controls. 100 μL of caspase 3/7 solution with buffer was then added to each of the wells and the cells were incubated for 1 h at 37°C in the dark. The fluorescence intensity was measured at Ex/Em=490/525 nm by Micro-plate Reader (Infinite F200, TEACH, Switzerland). 1.9 Single-pass Intestinal Perfusion Studies in Rats All intestinal perfusion studies were carried out in male Wistar rats (250±30 g). Animal studies were performed in accordance with a protocol approved by the Institutional Animal Experimentation Committee of Lanzhou University. Briefly, rat was fasted for 12 h and free accessed to water prior to this experiment. The single-pass intestinal perfusion was performed as described previously[26]. Before surgery, rats were anesthetized with an intraperitoneal injection of urethane (10 % w/v) at a dose of 10 mL/kg body weight. Then the rat was placed on a steady plate with a heating lamp above to maintain normal body temperature during the experiment. The abdomen was opened by a midline incision and a 10 cm segment of duodenum was isolated and cannulated with glass tubing. The intestinal segment was washed with intestinal perfusate maintained at 37°C for approximately 30 min until the outlet solution was visually clear. Then the intestinal segments were equilibrated with drug solution for 20 min and samples were quantitatively collected at 0, 15, 30, 45, 60, 75, 90 and 105 min. The tubes for sample collected were weighed before and

after each sample collection. The collected samples were filtered and then analyzed by HPLC. Finally, at the end of the experiment, the intestinal segments were removed and the length of the intestine was measured. The radius of the intestinal segment was 0.18 cm. The drug concentration in outflow perfusate was corrected against the water absorbance rate[26, 27]. The effective permeability (Peff) of a particular segment for each rat was calculated according to the formulas listed as follows: Peff=–Q•ln(Cout(corr)/ Cin•Vout/Vin)/2πrL, where Cin is the drug concentration in the inlet of the perfusate entering the intestinal segment, Cout(corr) is the corrected drug concentration, while Vin and Vout are their solute volumes, respectively, Q is the perfusion rate (0.2 mL/min), r is the radius of the intestinal segment, and L is the length of the intestinal segment. 1.10 Statistical Analysis All the experiments were performed in triplicate and results were expressed as ±s (n=3). Statistical analysis was done by performing Student’s t test. The differences were considered significant when P values were less than 0.05. 2 RESULTS AND DISCUSSION 2.1 Preparation and Characterization of Cur Nanoemulsions Cur nanoemulsions had a roughly spherical shape under the TEM (fig. 1A). The average particle size was 34.54±2.2 nm and the zeta potential value was approximately –8.54±0.45 mV. The polydispersity index of Cur nanoemulsions was 0.129±0.02, indicating a narrow size distribution (fig. 1B). Different from free Cur that was poorly soluble in water (fig. 2B), Cur nanoemulsions were a colloidal solution (fig. 2A), and were fully dispersible in water.

Fig. 1 General characteristics of the Cur nanoemulsions A: TEM images of Cur nanoemulsions; B: size distribution detected by dynamic light scattering

2.2 Cell Cytotoxicity Assay MTT assay showed that the cell cytotoxicity of free Cur and Cur nanoemulsions tended to increase with action time. Cur nanoemulsions, like free Cur, caused morphological changes in PC-3 cells (fig. 3). Cells treated with free Cur and Cur nanoemulsions turned spherical, whereas the untreated cells were still flat in shape. There was significant difference in cell viability between different free Cur groups (20–100 µmol/L) after 24-h treatment (fig. 4). However, no significant difference was noted in cell viability be-

tween 48 h and 72 h groups after treatment with free Cur at the same concentration. In contrast, Cur nanoemulsions could significantly inhibit the growth of PC-3 cells in a dose- and time-dependent manner, especially when the concentrations were above 20 µmol/L. The cell viability was 88.70%±2.10%, 71.80%±5.40%, and 69.75%±6.20% with 20 µmol/L free Cur at 24, 48 and 72 h, respectively. It was reduced to 74.95%±3.80%, 70.14%±6.40% and 22.21%±3.80% with Cur nanoemulsions at 24, 48 and 72 h, respectively. The effect was even more pro-

374 nounced with the concentration increasing.

Fig. 2 Enhancement of water solubility of Cur nanoemulsions A: Cur encapsulated in nanoemulsions is fully dispersible in water; B: Free Cur is poorly soluble in water and macroscopic flakes are visible especially on the bottom of the bottle.

The cell viability was changed to 78.70%±1.20%, 70.20%±4.7% and 68.94%±10.20% with 40 µmol/L free Cur at 24, 48 and 72 h, respectively, and it was reduced more significantly to 46.78%±2.30%,

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27.47±1.90% and 10.34±4.10% with 40 µmol/L Cur nanoemulsions. These findings suggested that Cur nanoemulsions could considerably reduce the cell viability as compared with free Cur, especially at 48 and 72 h. In our previous study the mean diameters of Cur nanoemulsions were measured at various time points for over three months of storage at 4°C[25]. The result indicated Cur nanoemulsions remained stable without any aggregation. The in vitro release of Cur nanoemulsions was also evaluated using a dialysis bag diffusion technique[25]. The results demonstrated that after 12-h incubation, only 30% of Cur was released from nanoemulsions at 37°C. Therefore, we speculated that the enhanced antiproliferation effect obtained by Cur nanoemulsions might be attributed to better stability of Cur with the protection of the carriers and the sustained release of Cur nanoemulsions. It was noted that at the highest concentration of 100 µmol/L, the free Cur only caused the lowest viability (approx 10%) for all treatment time. Further, we investigated the effect of RH40 and blank nanoemulsions on PC-3 cell line. The results showed that blank nanoemulsions except at the highest concentration had no significant effects on PC-3 cell viability (data not shown).

Fig. 3 Effect of Cur nanoemulsions on cell morphology A: Untreated PC-3 cells preserved their flat morphology throughout the study. B and C: Cells treated with free Cur (B) or Cur nanoemulsions (C) turned spherical after 48 h of exposure.

Fig. 4 Cell viability profile of PC-3 cells treated with free Cur and Cur nanoemulsions Cytotoxicity of Cur nanoemulsions and free Cur (in DMSO) to PC-3 cells was investigated at various concentrations for 24, 48 and 72 h. Cell viabilities are given in percentages relative to untreated cells.

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2.3 Cell Uptake Results There was significant difference in Cur uptake by PC-3 cells between free Cur and Cur nanoemulsions groups (fig. 5A). Free Cur could rapidly accumulate to the maximum level in the cytosol within the incubation time (up to 1 h), while Cur nanoemulsions reached the maximum level over a 3-h incubation. The drug levels in cells treated with free Cur and Cur nanoemulsions for 1 h were 2.82±0.11 and 1.52±0.14 µg per 105 cells, respectively. After that, the drug levels were reduced significantly in free Cur-treated cells compared to Cur nanoemulsion-treated cells. At 3 h, the drug levels were reduced to 1.49±0.12 µg per 105 cells with free Cur, while the highest drug levels of Cur nanoemulsions (1.73±0.15 µg per 105 cells) were observed. Significant reduction in drug levels was observed after 3 h in free Cur-treated cells compared with Cur nanoemulsion-treated cells. At the end of the experiment (24 h), the reduction in drug levels of free Cur-treated cells was more significant (0.22±0.069 µg per 105 cells) than that in Cur nanoemulsion-treated cells (1.47±0.11 µg per 105 cells). Cells treated with Cur nanoemulsions exhibited slightly decreased fluorescence intensity at 24 h. The reduction in fluorescence intensity with time in cells treated with free Cur suggested the instability of Cur with time. However, the maximum uptake of free Cur (dissolved in DMSO) was 1.7 fold higher than that of Cur nanoemulsions. Because of the lipophilicity of Cur (log P=3.67), we assumed that Cur could be easily transported passively in a form of solubilised molecule. The results did demonstrate that Cur could accumulate in

375 PC-3 cells within a short time,. Previous studies reported that solubilised Cur permeated across the monolayers rapidly [P(app)(A−B)=(7.1±0.7)×10-6 cm/s] by passive diffusion[28]. The t1/2s for the degradation of Cur is 9 min (pH 7.2) and 199 min (pH 5) in buffer solutions at 37°C[29]. Cur within the cells degraded rapidly because it was unstable in the culture medium. Cur alone has no statistically significant effect on cell survival or growth due to its very poor solubility in water[24]. As a consequence, Cur delivered as the nanoemulsion could enhance its stability and gradually release due to the incorporation into the nanocarrier. Our results also indicated that the present formulation could successfully prevent the hydrolysis of Cur. The mechanism of cellular uptake of the nanoparticles was not investigated in this study, and we speculated that they most likely entered the cells by endocytosis. The results outlined above suggest that nanosizing may not always ensure better cellular uptake than the solubilised solution. In this study, fluorescence microscopy of free Cur and Cur nanoemulsions also revealed enhanced and sustained uptake of Cur nanoemulsions compared with free Cur (fig. 5B). Moreover, MTT assay demonstrated the growth inhibition of PC-3 cells in a time-dependent manner was more obvious in Cur nanoemulsions than in free Cur. These results further corroborated previous studies which also revealed that Cur nanoemulsions could enhance the anti-cancer capacities of Cur by increasing cell uptake, sustaining drug release and increasing drug accumulation[16].

Fig. 5 Cell uptake results A: HPLC analysis of cell uptake of free Cur and Cur nanoemulsions by PC-3 cells; B: fluorescence microscopy images showing the time-dependent (1, 3, and 12 h) intracellular uptake of free Cur and Cur nanoemulsions by PC-3 cells. Cells were treated with 60 µmol/L free Cur or Cur nanoemulsions. Data were expressed as ±s (n=3). *P<0.01, Cur nanoemulsion vs. free Cur.

2.4 Cell Cycle Results Consistent with the previous study[30], as shown in fig. 6, flow cytometry demonstrated that PC-3 cells exposed to free Cur (60 μmol/L) for 24 h were different from untreated ones in terms of a higher population in the G2/M phase (32.74% vs. 16.9%) and fewer fractions in the G0/G1 phase (42.58% vs. 64.7%; fig. 6). These results indicated that PC-3 cell cycle was arrested in G2/M phase by free Cur. Similar to the results for PC-3 cell uptake, G2/M phase arrest in free Cur group declined obviously after reaching a peak. The cell population in G2/M phase was declined from 19.53% to 9.81%, as the cell population in G0/G1

phase changed from 57.71% to 81.23% from 48 to 72 h, respectively. On the contrary, Cur nanoemulsion treatment at 60 μmol/L resulted in a relatively steady state of cell population in G2/M phase from 30.57% to 31.37% and then to 23.68%, as cell population in G0/G1 phase changed from 52.98% to 53.42% and then to 61.93%, from 24 to 48 h and subsequently to 72 h, respectively. Cell cycle profiles of untreated cells remained unaltered throughout the experiment. Collectively, different from free Cur, Cur nanoemulsions induced more PC-3 cells arrested in G2/M phase, which might be related to the slow release of nanoemulsions[16].

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Fig. 6 A: Effect of free Cur and Cur nanoemulsions on cell cycle by flow cytometry at 24, 48 and 72 h; B: The histogram plot represents the distribution of cells in sub G0/G1, S and G2/M phase.

2.5 Apoptosis Analysis by Caspase 3/7 Activity Fig. 7 shows the caspase 3/7 activity in PC-3 cells following treatment with free Cur and Cur nanoemulsions. The caspase activity was increased by 1.5- to 3.1-fold in Cur nanoemulsion group relative to control group. In particular, at 72 h Cur nanoemulsions showed 3.1-fold higher caspase activity while the activity was 2.4-fold higher in free cur group than in

control group. The results revealed the apoptosis was significantly increased in PC-3 cells treated with Cur nanoemulsions when compared with free Cur. These results strongly suggested that Cur nanoemulsions caused increased cell cytotoxicity which might be attributed to increased cellular apoptosis and G2/M phase cell cycle arrest.

Fig. 7 The caspase 3/7 activity in PC-3 cells after treatment with free Cur and Cur nanoemulsions

2.6 Intestinal Absorption of Cur Nanoemulsions The Peff of Cur nanoemulsions was (1.80±0.48)×10-3, (1.59±0.90)×10-3, (1.12±0.35)×10-3 cm/min, in the duodenum, jejunum and ileum respectively (fig. 8), which was 1.4-fold and 1.67-fold higher than that of free Cur (in DMSO) in the duodenum and jejunum, respectively (P<0.05). The increase of Peff in Cur nanoemulsions indicated the potential of nanoemulsion improving the oral delivery of Cur. Previous studies reported that nanoemulsions could significantly increase the oral absorption of Cur compared with its suspension[20, 22]. The relative bioavailability was found to be 26.45-fold higher with nanoemulsions than that obtained after the administration of single oral dose of Cur suspension[22]. Our findings were slightly different from theirs, which might be due to the Cur solution (in DMSO) used as the control in our study, instead of Cur suspension.

According to previously reported mechanisms[31–33], the enhanced absorption of nanoemulsions may be attributed to the improvement in the solubilization and the increase in the membrane fluidity by surfactant components. The high content of surfactants in nanoemulsion formulations could increase the permeability by disturbing the cell membrane, the single layer of intestinal epithelial cell as the main barrier for drug absorption[24]. RH40 was known to modulate the membrane fluidity and used as a vehicle for solubilization of hydrophilic drugs[32]. In this study, RH40 as a surfactant was chosen in the formulations to improve the solubility and oral absorption of Cur. In addition, lymphatic transport was demonstrated to contribute to the bioavailability of the oral nanoformulations[20, 26]. Cur nanoemulsions affecting the absorption in the intestine are possibly related to the mechanism mentioned above, which needs further studies.

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Fig. 8 The permeability coefficients of Cur nanoemulsions in different intestinal segments (n=4) * P<0.05, **P<0.01 Cur nanoemulsions vs. free Cur

To sum up, the present study corroborated that nanoemulsions could be loaded efficiently with Cur and increase the solubilization of the encapsulated drug. Cur-based nanoemulsions could enhance the cellular cytotoxicity, cellular uptake, cell cycle arrest and apoptosis against prostate cancer cells. Cur encapsulated was gradually released into the cells, thereby resulting in prolonged cytotoxicity and cell cycle arrest in PC-3 cells. In situ single-pass perfusion studies demonstrated higher effective permeability coefficient for Cur nanoemulsions than for free Cur. Thus, these results suggested that Cur nanoemulsions have superior anticancer potential than the same dose of free Cur. Our study provides the evidence for the therapeutic applications of Cur nanoemulsions and an alternative platform for the delivery of hydrophobic bioactive agents in future clinical studies. Conflict of Interest Statement The authors declare no conflicts of interest in relation to this work. REFERENCES 1 Anand P, Kunnumakkara AB, Newman RA, et al. Bioavailability of curcumin: problems and promises. Mol Pharm, 2007,4(6):807-818 2 Sun J, Bi C, Chan HM, et al. Curcumin-loaded solid lipid nanoparticles have prolonged in vitro antitumour activity, cellular uptake and improved in vivo bioavailability. Colloids Surf B Biointerfaces, 2013,111(11): 367-375 3 Kanai M, Otsuka Y, Otsuka K, et al. A phase I study investigating the safety and pharmacokinetics of highly bioavailable curcumin (Theracurmin) in cancer patients. Cancer Chemother Pharmacol, 2013,71(6):1521-1530 4 Singla V, Mouli VP, Garg SK, et al. Induction with NCB-02 (curcumin) enema for mild-to-moderate distal ulcerative colitis---A randomized, placebo-controlled, pilot study. J Crohns Colitis, 2014,8(3):208-214 5 Nabavi, SF, Daglia M, Moghaddam AH, et al. Curcumin and liver disease: from chemistry to medicine. Compr Rev Food Sci F, 2014,13(1):62-77 6 Teiten MH, Gaascht F, Eifes S, et al. Chemopreventive potential of curcumin in prostate cancer. Genes Nutr, 2010,5(1):61-74

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