A Novel Solubility-Enhanced Rubusoside-Based Micelles for Increased Cancer Therapy
© The Author(s). 2017
Received: 1 March 2017
Accepted: 6 April 2017
Published: 13 April 2017
Many anti-cancer drugs have a common problem of poor solubility. Increasing the solubility of the drugs is very important for its clinical applications. In the present study, we revealed that the solubility of insoluble drugs was significantly enhanced by adding rubusoside (RUB). Further, it was demonstrated that RUB could form micelles, which was well characterized by Langmuir monolayer investigation, transmission electron microscopy, atomic-force microscopy, and cryogenic transmission electron microscopy. The RUB micelles were ellipsoid with the horizontal distance of ~25 nm and vertical distance of ~1.2 nm. Insoluble synergistic anti-cancer drugs including curcumin and resveratrol were loaded in RUB to form anti-cancer micelles RUB/CUR + RES. MTT assay showed that RUB/CUR + RES micelles had more significant toxicity on MCF-7 cells compared to RUB/CUR micelles + RUB/RES micelles. More importantly, it was confirmed that RUB could load other two insoluble drugs together for remarkably enhanced anti-cancer effect compared to that of RUB/one drug + RUB/another drug. Overall, we concluded that RUB-based micelles could efficiently load insoluble drugs for enhanced anti-cancer effect.
KeywordsRubusoside Poor solubility Curcumin Resveratrol Cancer
Although chemotherapy is one of the most commonly used approaches to treat cancer, conventional chemotherapeutics usually led to numerous unfavorable side effects owing to insolubility, multi-drug resistance, and poor selectivity towards cancer cells [1, 2].
Combination therapy usually resulted in survival advantage over monotherapy, which had become a common approach for the treatment of most cancer. Both CUR and RES could act as inducers of chromosomal aberrations leading to cell death or apoptosis in cancer cell lines [10–13]. They could synergistically cause apoptosis in breast cancer cells induced by cigarette smoke . Although the safety and efficacy of CUR and RES synergistically against some diseases had been reported, their further application had been limited owing to poor solubility . So, enhancing their solubility was much essential for their pharmaceutical applications.
Rubusoside (RUB; Fig. 1c) is a diterpene glycoside mainly from Chinese sweet leaf tea leaves (Rubus suavissimus; Rosaceae) . It was a well-known natural sweetening agent and had been used in food and beverage products. Recently, RUB has been increasingly attracting attention for its solubilizing properties [17–20]. However, the solubilization mechanism of RUB is still unclear until now.
In this study, RUB was used as a solubilizer for CUR and RES solubilization, the solubilization mechanism was investigated with Langmuir monolayer measurement, TEM, cryo-TEM, and AFM. It was demonstrated that RUB could form micelles. Further, the synergistic anti-cancer effects of CUR and RES in different RUB-based micelle formulations were determined on MCF-7 cells.
Rubusoside, curcumin, and resveratrol were obtained from Shanghai Qiaoyu Company. Dulbecco’s modified Eagle’s medium (DMEM; high glucose), fetal bovine serum (FBS; Australian origin), penicillin and streptomycin, and EDTA solution (0.25% trypsin with 0.53 mM EDTA) were purchased from Life Technologies (Grand Island, NY, USA). MTT reagent was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were analyzed by HPLC or of analytical grade.
High-Performance Liquid Chromatography (HPLC) Measurement
A HPLC system (1260, Agilent, USA) was used for the analyses. All of the analyses were carried out on a Diamonsil ODS C18 HPLC column at 25 °C. For the detection of RUB, elution was performed with acetonitrile and water (v/v, 33:67). For CUR detection, elution was conducted with methanol and 3.6% acetic acid (v/v, 75:25). For RES detection, elution was completed with acetonitrile and 0.5% acetic acid (v/v, 54:46). For ginsenoside (Rh2) (Additional file 1: Figure S1a) detection, elution was performed with acetonitrile and water (v/v, 70:30). For silymarin (SM) (Additional file 1: Figure S1b) detection, elution was set with methanol/acetonitrile/1% acetic acid acetonitrile (v/v, 40.4:9.6:50). The flow rate of the mobile phase was 1.0 ml/min and the injection volume of the sample was 20 μl. The selected detection wavelength was 426 nm for CUR, 215 nm for RUB, 306 nm for RES, 203 nm for Rh2, and 288 nm for SM. Each sample was subjected to a final step of filtration with a 0.45-μm nylon filter before injection.
Preparation of RUB-Based Micelles
The appropriate amounts of RUB, CUR, and RES were added into a bottle and vortexed slightly to form a suspension solution. The emulsion was then subjected to an autoclave at 121 °C and 0.11 MPa for 60 min. Samples after heating in the autoclave were supersaturated solutions showing CUR and RES partial precipitation. Then, it was kept in an incubator at 25 °C for 12 h to equilibrate. Finally, each was subjected to a final step of filtration with a 0.45-μm cellulose membrane filter. All products were protected from light and kept at room temperature.
Langmuir Monolayer Measurement
The RUB formulations were dissolved in chloroform/methanol (v/v, 9:1) and be deposited onto the subphase of the minitrough (KSV, Finland) using a microsyringe. Compression was initiated to allow the solvent evaporation. The compression rate was 10 mm/min. Surface pressure-molecular area (π-A) isotherms were determined and processed with the Layer-Builder Analysis Software (KSV). The experiments were performed at 25 °C.
Characterization of RUB-Based Micelles
Surface morphology of RUB-based micelles was measured by transmission electron microscopy (JEM-2100, 200 kV). Vitrified specimens for cryogenic transmission electron microscopy (Cryo-TEM; Tecnal G20)  imaging were in a controlled environment vitrification system (CEVS) at 25 °C and 100% relative humidity. The morphology  of the RUB-based micelles was further examined using atomic-force microscopy (AFM; E-Sweep, Seiko, Japan). The sample was prepared by placing a drop onto mica (Asheville-Schoonmaker Mica Co, Newport News, VA). Subsequently, the sample was imaged by scanning 1 μm × 1 μm areas in tapping mode using an OMCL-AC160TS cantilever with 115–190 kHz resonance frequencies and a constant force ranging from 2.5–10 N/m. The size and zeta potential of the preparations were investigated with a Nano ZS90 Zetasizer (Malvern Instruments Ltd., Malvern, UK). The phase transition process of RUB-based micelles was performed using differential scanning calorimetry (NETZSCH Gerätebau GmbH, Selb, Germany) at a heating rate of 10 °C/min from 30 to 250 °C. X-ray diffraction analysis (XRD; D8 Advance, Bruker, Germany) was applied to further investigate the physical state of RUB-based micelles.
Critical Micelle Concentration (CMC) Measurement
The CMC of RUB micelles were measured by fluorescence measurement using pyrene as a probe [23–25]. The fluorescence emission spectra of pyrene (6 × 10−7 M) in different concentrations (varying from 0.01 to 0.4 mM) of RUB solution were determined using a fluorescence spectrophotometer, with the excitation wavelength of 335 nm. The intensities of I3 (394 nm) and I1 (378 nm) were measured at the wavelengths corresponding to the third and first highest energy bands. Then, the intensity ratio of I3 to I1 (I3/I1) in the pyrene emission spectra was calculated.
In Vitro Drug Release
The drug (CUR and RES) release from RUB/CUR + RES micelles and RUB/RES micelles + RUB/CUR micelles were investigated by a dialysis method in 100 ml of phosphate-buffered saline (PBS; pH 7.4) containing 0.5% Tween 80 at 37 °C . Two milliliters of the sample was placed in a dialysis bag (molecular weight cutoff, 14,000). The bag was then tied and immersed in medium in a shaker bath (100 strokes/min). At a defined time interval, 100 μl of the sample was withdrawn and replaced with the same volume of fresh medium. The drug concentration was measured by HPLC.
MCF-7 cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). They were cultured at 37 °C under 5% CO2 in DMEM supplemented with 10% fetal bovine serum (PAA, Austria). The cultured cells were trypsinized once 80% confluence with 0.25% trypsin-EDTA solution (Sigma, USA).
Cell Apoptosis Measurement
Annexin V-FITC/PI dual staining for apoptosis was performed to measure the apoptosis. Briefly, 1 × 105 cells/well of MCF-7 cells were seeded in a six-well cell culture plate and incubated for 24 h. Subsequently, cells were exposed to free RES + CUR, RUB/CUR + RES micelles, or RUB/RES micelles + RUB/CUR micelles. Cells were harvested post-incubation by trypsinization and washed twice with PBS. Then, cells were resuspended in binding buffer and stained with Annexin V-FITC and PI detection kit according to the protocol provided by the manufacturer. Stained cells were analyzed by a FACS Aria II flow cytometer.
Anti-Cancer Effect of RUB-Based Micelles
The anti-cancer effect of RUB-based micelles on MCF-7 cells was assessed via MTT method. Cells (1 × 105 cells/ml) were cultured overnight in 96-well plates. Subsequently, they were treated with RES + CUR, RUB/CUR + RES micelles or RUB/RES micelles + RUB/CUR micelles at the concentration of CUR (23 μM) and RES (110 μM) for 24 h. Control cells were treated with PBS. At indicated time points, 10 μl MTT (5 mg/ml) was added and plates were incubated at room temperature for another 4 h in the dark. Then, the medium was replaced with 150 μl DMSO, and plates were further incubated for 10 min. OD570nm was measured using a microplate reader (Bio-Rad Laboratories Inc, Hercules, CA, USA).
Cellular Localization of RUB-Based Micelles in MCF-7 Cells
To image the intracellular localization of the micelles, MCF-7 cells were incubated with free coumarin-6 (C6) or C6-loaded micelles (C6 content, 4.5 mg/ml) for 15 min, 1 h, and 2 h at 37 °C. After that, the culture medium was aspirated and cells were washed three times with PBS, followed by cell fixation with 4% paraformaldehyde. Next, the nucleus was stained with DAPI for 10 min at 37 °C. The fluorescence was then visualized using a confocal laser scanning microscope (LSM710, Zeiss, Germany).
Cellular Uptake by Flow Cytometry
MCF-7 cells were seeded on six-well culture plates (1 × 105 cells/well) and incubated for 24 h in DMEM medium containing 10% FBS. Subsequently, they were treated with free coumarin-6 (C6) or C6-loaded micelles (with an equivalent C6 concentration of 4.5 mg/ml). After incubation, they were harvested and suspended in 500 μl of PBS. The cellular uptake of micelles was determined using a flow cytometry (Becton Dickinson, FACS, Aria II).
All data were represented as mean ± SD from at least three independent experiments. Statistical significance analysis was conducted using Student’s t test. P < 0.05 was considered statistically significant.
Results and Discussion
Characterization of the RUB-Based Nanoparticles
As outlined in Fig. 1b, c, RUB and RUB/CUR + RES could form self-assembled nanoparticles in aqueous condition. It was proved that RUB could form round nanoparticles of about 25 nm in diameter by TEM (Fig. 2b). Cryo-TEM result indicated that the morphology of nanoparticles was round with hollow (Fig. 2c) and its measured size is also ~25 nm. AFM measurement further verified that RUB-formed nanoparticles were ellipsoid with the horizontal distance of ~25 nm and vertical distance of ~1.2 nm (Fig. 2d and Additional file 1: Figure S2).
The Critical Micelle Concentrations (CMC) of RUB Micelles
Size and Zeta Potential of RUB-Based Micelles
The particle size and surface zeta potential of RUB micelles, RUB/CUR + RES micelles, and RUB/CUR micelles + RUB/RES micelles are shown in Fig. 3c, d, which was measured by DLS. Micelles with smaller size tended to accumulate easily in tumor sites due to the enhanced permeability and retention (EPR) effect and gained a faster internalization rate into cells [30, 31]. Results showed that the sizes of RUB-based micelles were all small micelles in different ways (Fig. 3c), which might benefit from well hydrophobic interaction between the hydrophobic cores of RUB micelles and insoluble drugs. The three kinds of RUB-based micelles had a similar zeta potential value because of their similar surface characterization (Fig. 3d).
X-Ray Diffraction (XRD) Measurement
Differential Scanning Calorimeter (DSC) Analysis
DSC measurements were used to acquire information on the crystallinity and polymorphism of the interaction between the drug and micelles from DSC thermograms. It could provide information including the appearance of new peaks, the elimination of endothermic peaks, and changes in peak shape and onset, peak temperature, or enthalpy . As shown in Fig. 4b, the thermograms of the physical mixture of raw materials showed three peaks at 131.9, 179.2, and 266.1 °C, which well corresponded to RUB power’s peak at 135.6 °C, CUR power’s peak at 181.2 °C, and RES power’s peak at 269.8 °C. The peaks of RES power and CUR power disappeared when they were loaded in RUB micelles to form RUB/CUR + RES micelles, which indicated that raw materials lost its crystallinity [33, 34].
Cell Uptake of RUB-Based Micelles in MCF-7 Cells
In Vitro Anti-Cancer Effect of RUB-Based Micelles
Here, cytotoxicity of blank RUB micelles on MCF-7 cells was evaluated. It demonstrated that high cell viabilities in blank RUB micelles at a concentration of 4–32 mmol/l and declined cell viabilities (at a concentration of above 32 mmol/l) in MCF-7 cells (Additional file 1: Figure S4), indicating that blank RUB micelles does not bring significant additional toxicity to cells at a concentration of below 32 mmol/l. This result confirmed blank RUB micelles are biocompatible.
Based on our results that RUB micelles loaded two insoluble drugs together had remarkably enhanced anti-cancer effect than that of RUB/one drug + RUB/another drug, it is highly interesting to explore the possibility in extending RUB-based micelles to other two insoluble drug encapsulation. Previous reports have revealed that ginsenoside (Rh2) and silymarin (SM) had therapeutic effects for some types of cancer and were insoluble [35, 36].
In this study, it was proved that RUB was self-assembled to form micelles. The RUB-based micelle system developed in this study was a promising small molecule carrier that efficiently improved the solubility of insoluble drugs. CUR and RES were loaded in RUB to form anti-cancer micelles RUB/CUR + RES. Interestingly, RUB/CUR + RES micelles had more remarkable toxicity on MCF-7 cells compared to RUB/CUR micelles + RUB/RES micelles. More importantly, it was proved that RUB could load other two insoluble drugs together for remarkably enhanced anti-cancer effect compared to that of RUB/one drug + RUB/another drug. Overall, RUB-based micelles could efficiently load insoluble anti-cancer drugs for significantly enhanced anti-cancer effect.
MYZ and TCD were actively involved in all the material and biological experiments. MYZ and TCD have written the manuscript. NPF designed all the experiments. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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- Kedar U, Phutane P, Shidhaye S, Kadam V (2010) Advances in polymeric micelles for drug delivery and tumor targeting. Nanomedicine 6:714–29View ArticleGoogle Scholar
- Ng KE, Amin MC, Katas H, Amjad MW, Butt AM, Kesharwani P et al (2016) pH-responsive triblock copolymeric micelles decorated with a cell-penetrating peptide provide efficient doxorubicin delivery. Nanoscale Res Lett 11:539–52View ArticleGoogle Scholar
- Araujo CC, Leon LL (2001) Biological activities of Curcuma longa L. Mem Inst Oswaldo Cruz 96:723–8View ArticleGoogle Scholar
- Jayakumar V, Ahmed SS, Ebenezar KK (2016) Multivariate analysis and molecular interaction of curcumin with PPARγ in high fructose diet induced insulin resistance in rats. Springer Plus 5:1732–47View ArticleGoogle Scholar
- Li SY, Wang XB, Kong LY (2014) Design, synthesis and biological evaluation of imine resveratrol derivatives as multi-targeted agents against Alzheimer’s disease. Eur J Med Chem 71:36–45View ArticleGoogle Scholar
- Fibach E, Prus E, Bianchi N, Zuccato C, Breveglieri G, Salvatori F et al (2012) Resveratrol: antioxidant activity and induction of fetal hemoglobin in erythroid cells from normal donors and beta-thalassemia patients. Int J Mol Med 29:974–82Google Scholar
- Csuk R, Albert S, Siewert B, Schwarz S (2012) Synthesis and biological evaluation of novel (E) stilbene-based antitumor agents. Eur J Med Chem 54:669–78View ArticleGoogle Scholar
- Androutsopoulos VP, Ruparelia KC, Papakyriakou A, Filippakis H, Tsatsakis AM, Spandidos DA (2011) Anticancer effects of the metabolic products of the resveratrol analogue, DMU-212: structural requirements for potency. Eur J Med Chem 46:2586–95View ArticleGoogle Scholar
- Biagi M, Bertelli AA (2015) Wine, alcohol and pills: what future for the French paradox? Life Sci 131:19–22View ArticleGoogle Scholar
- Goel A, Aggarwal BB (2010) Curcumin, the golden spice from Indian saffron, is a chemosensitizer and radiosensitizer for tumors and chemoprotector and radioprotector for normal organs. Nutr Cancer 62:919–30View ArticleGoogle Scholar
- Zoberi I, Bradbury CM, Curry HA, Bisht KS, Goswami PC, Roti Roti JL et al (2002) Radiosensitizing and anti-proliferative effects of resveratrol in two human cervical tumor cell lines. Cancer Lett 175:165–73View ArticleGoogle Scholar
- Veeraraghavan J, Natarajan M, Lagisetty P, Awasthi V, Herman TS, Aravindan N (2011) Impact of curcumin, raspberry extract, and neem leaf extract on rel protein-regulated cell death/radiosensitization in pancreatic cancer cells. Pancreas 40:1107–19View ArticleGoogle Scholar
- Fang Y, Demarco VG, Nicholl MB (2012) Resveratrol enhances radiation sensitivity in prostate cancer by inhibiting cell proliferation and promoting cell senescence and apoptosis. Cancer Sci 103:1090–8View ArticleGoogle Scholar
- Mohapatra P, Satapathy SR, Siddharth S, Das D, Nayak A, Kundu CN (2015) Resveratrol and curcumin synergistically induces apoptosis in cigarette smoke condensate transformed breast epithelial cells through a p21 (Waf1/Cip1) mediated inhibition of Hh-Gli signaling. Int J Biochem Cell Biol 66:75–84View ArticleGoogle Scholar
- Bisht K, Wagner KH, Bulmer AC (2010) Curcumin, resveratrol and flavonoids as anti-inflammatory, cyto- and DNA-protective dietary compounds. Toxicology 278:88–100View ArticleGoogle Scholar
- Liu, Z (2009) Diterpene glycosides as natural solubilizers. US Patent Application PCT/US 040324Google Scholar
- Jeansonne DP, Koh GY, Zhang F, Kirk-Ballard H, Wolff L, Liu D et al (2011) Paclitaxel-induced apoptosis is blocked by camptothecin in human breast and pancreatic cancer cells. Oncol Rep 25:1473–80Google Scholar
- Zhang F, Koh GY, Hollingsworth J, Russo PS, Stout RW, Liu Z (2012) Reformulation of etoposide with solubility-enhancing rubusoside. Int J Pharm 434:453–9View ArticleGoogle Scholar
- Zhang F, Koh GY, Jeansonne DP, Hollingsworth J, Russo PS, Vicente G et al (2011) A novel solubility-enhanced curcumin formulation showing stability and maintenance of anticancer activity. J Pharm Sci 100:2778–89View ArticleGoogle Scholar
- Liu Z, Zhang F, Koh GY, Dong X, Hollingsworth J, Zhang J et al (2015) Cytotoxic and antiangiogenic paclitaxel solubilized and permeation-enhanced by natural product nanoparticles. Anticancer Drugs 26:167–79View ArticleGoogle Scholar
- Yang X, Koh CG, Liu S, Pan X, Santhanam R, Yu B et al (2009) Transferrin receptor-targeted lipid nanoparticles for delivery of an antisense oligodeoxyribonucleotide against Bcl-2. Mol Pharm 6:221–30View ArticleGoogle Scholar
- Chen Z, Wang Z, Chen J, Chen X, Wu J, Wu Y et al (2013) Resonance light scattering technique as a new tool to determine the binding mode of anticancer drug oridonin to DNA. Eur J Med Chem 66:380–7View ArticleGoogle Scholar
- You J, Hu FQ, Du YZ, Yuan H (2007) Polymeric micelles with glycolipid-like structure and multiple hydrophobic domains for mediating molecular target delivery of paclitaxel. Biomacromolecules 8:2450–6View ArticleGoogle Scholar
- Molina-Bolívar JA, Hierrezuelo JM, Carnero Ruiz C (2007) Self-assembly, hydration, and structures in N-decanoyl-N-methylglucamide aqueous solutions: effect of salt addition and temperature. J Colloid Interface Sci 313:656–64View ArticleGoogle Scholar
- Cai LL, Liu P, Li X, Huang X, Ye YQ, Chen FY et al (2011) RGD peptide-mediated chitosan-based polymeric micelles targeting delivery for integrin-overexpressing tumor cells. Int J Nanomedicine 6:3499–508Google Scholar
- Liu Y, Wang P, Sun C, Feng N, Zhou W, Yang Y et al (2010) Wheat germ agglutinin-grafted lipid nanoparticles: preparation and in vitro evaluation of the association with Caco-2 monolayers. Int J Pharm 397:155–63View ArticleGoogle Scholar
- Jin Y, Lian Y, Du L, Wang S, Su C, Gao C (2012) Self-assembled drug delivery systems. Part 6: in vitro/in vivo studies of anticancer N-octadecanoyl gemcitabine nanoassemblies. Int J Pharm 430:276–81View ArticleGoogle Scholar
- Korchowiec B, Paluch M, Corvis Y, Rogalska E (2006) A Langmuir film approach to elucidating interactions in lipid membranes: 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine/cholesterol/metal cation systems. Chem Phys Lipids 144:127–36View ArticleGoogle Scholar
- Chen W, Chen HR, Hu JH, Yang WL, Wang CC (2006) Synthesis and characterization of polyion complex micelles between poly (ethyleneglycol)-grafted poly (aspartic acid) and cetyltrimethyl ammonium bromide. Colloids Surf A Physicochem Eng Asp 278:60–66View ArticleGoogle Scholar
- Maeda H (2001) The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul 41:189–207View ArticleGoogle Scholar
- Maeda H, Wu J, Sawa T, Matsumura Y, Hori K (2000) Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release 65:271–84View ArticleGoogle Scholar
- Bunjes H, Unruh T (2007) Characterization of lipid nanoparticles by differential scanning calorimetry, X-ray and neutron scattering. Adv Drug Deliv Rev 59:379–402View ArticleGoogle Scholar
- Chen L, Sha X, Jiang X, Chen Y, Ren Q, Fang X (2013) Pluronic P105/F127 mixed micelles for the delivery of docetaxel against Taxol-resistant non-small cell lung cancer: optimization and in vitro, in vivo evaluation. Int J Nanomedicine 8:73–84Google Scholar
- Hu Y, Xie J, Tong YW, Wang CH (2007) Effect of PEG conformation and particle size on the cellular uptake efficiency of nanoparticles with the HepG2 cells. J Control Release 118:7–17View ArticleGoogle Scholar
- Li L, Sun HY, Liu W, Zhao HY, Shao ML (2017) Silymarin protects against acrylamide-induced neurotoxicity via Nrf2 signalling in PC12 cells. Food Chem Toxicol 102:93–101View ArticleGoogle Scholar
- Kim JH, Choi JS (2016) Effect of ginsenoside Rh-2 via activation of caspase-3 and Bcl-2-insensitive pathway in ovarian cancer cells. Physiol Res 65:1031–7Google Scholar