http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–9 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.920936
RESEARCH ARTICLE
Preparation, characterization, and anticancer efficacy of evodiamine-loaded PLGA nanoparticles Lidi Zou1*, Fengqian Chen1*, Jiaolin Bao1, Shengpeng Wang1, Lu Wang1, Meiwan Chen1,2, Chengwei He1, and Yitao Wang1 1
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China and Stake Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China
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Abstract
Keywords
Evodiamine (EVO) is a plant-derived indolequinazoline alkaloid with potential anticancer activity. However, low bioavailability caused by its poor water solubility limits it anticancer efficacy in clinic. To enhance the solubility and improve the bioavailability of EVO, a delivery system based on poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with EVO (EVO-PLGA NPs) for treating breast cancer was prepared in this study. The physicochemical characterization and in vitro antitumor evaluation of EVO-PLGA NPs were determined. EVO-PLGA NPs could persistently control the release of EVO for 180 h. 3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) assessment and colony formation assay showed that EVO-PLGA NPs could enhance the toxicity and the proliferation inhibition effect of EVO on MCF-7 breast cancer cells. EVO-PLGA NPs did not strengthen G2/M arrest effect of EVO-treated cells after 24h incubation. Meanwhile, EVO-PLGA NPs could increase the expression of cyclin B1 and decrease the expression of b-actin. Taken together, these results suggested that -PLGA NPs is promising for improving anticancer efficacy of EVO in breast cancer therapy.
Anticancer activity, breast cancer, evodiamine, formulation, PLGA nanoparticles
Introduction Breast cancer is one of the most prevalent malignancies in women diseases (Beral et al., 2003). However, numerous anticancer drugs have high toxicity, which leads to side effects, and have poor solubility that could decrease the drug efficacy. One approach to overcome these limitations is to develop a reliable nanoparticles (NPs) delivery strategies, which possesses several unique advantages, such as largely increased the aqueous solubility and improved bioavailability of the anticancer drugs, elongated the circulation time of blood, enhanced permeability and retention (EPR) so as to enhance the accumulation in the tumor sites and reduced systemic side effects (Torchilin, 2011). Thus, it is important to develop NPs delivery systems for anticancer agents. Evodiamine (EVO), an indolequinazoline alkaloid, is widely found in many medicinal plants of the tetradium family (Dong et al., 2012; Schwarz et al., 2013). It has been demonstrated that EVO possessed a plenty of pharmacologic activities, such as anti-obesity, anti-inflammatory (Chen et al., 2012), and anti-infectious effect (Li-Weber, 2013; Yu et al., 2013). Furthermore, many studies *These authors contributed equally to this work. Address for correspondence: Dr Meiwan Chen and Dr Chengwei He, State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Av. Padre Tomas Pereira S.J., Taipa, Macau, China. E-mail: [email protected] (M. Chen) and [email protected] (C. He)
History Received 14 March 2014 Revised 30 April 2014 Accepted 30 April 2014
declared that EVO was a promising anti-tumor agent (Jiang & Hu, 2009), which could suppress proliferation (Chen et al., 2010), induce apoptosis (Rasul et al., 2012), and inhibit metastasis (Takada et al., 2005) of cancer cells. These capabilities were believed to function well through the effects of EVO, which could alter the balance of Bcl2 and Bax gene expression by the caspase pathway in cancer cells (Fei et al., 2003). Furthermore, EVO could not only inhibit signal transducer and activator of transcription-3 (STAT3) DNAbinding activity, but also down-regulate the expression of STAT3-mediated genes leading to the suppression of proliferation and induction of cell apoptosis and cell cycle arrest (Yang et al., 2012). EVO, one of dissociative alkaloid in nature, possesses poor solubility in aqueous solution so as to limit EVO in systemic bioavailability and clinical efficacy (Tan et al., 2012; Xu et al., 2012). To increase the solubility of EVO, a NPs delivery system based on poly (lactic-co-glycolic acid) (PLGA) were prepared by a single emulsion (o/w) solvent evaporation technique (Manchanda et al., 2010). PLGA, which is approved by European Medicine Agency as non-toxic materials to prepare nanoparticle drug delivery systems (Danhier et al., 2012), was chosen to be the delivery material in this study for its well biodegradability property (Acharya & Sahoo, 2011; Anderson & Shive, 1997) and its ability of controlling sustained release (Chung et al., 2010). The EVO-loaded PLGA NPs (EVO-PLGA NPs) were developed to investigate the proliferation inhibition effect of breast cancer MCF-7 cells, aiming
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to disclose the relevant factors related to the pharmacologic activity of EVO-loaded PLGA NPs. EVO-PLGA NPs were characterized by its encapsulation efficiency, particle size, zeta potential, in vitro drug release behavior, and anticancer activity to MCF-7 human breast adenocarcinoma cell line. These results suggested that the application of EVO-PLGA NPs delivery system is promising for improvement of anticancer efficacy in cancer therapy.
Materials and methods
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Materials EVO was obtained from Chengdu Must Bio-Technology Co., Ltd, and the purity was determined to be 98% by highperformance liquid chromatography (HPLC). PLGA with different molecular weights and polyvinyl alcohol (PVA, molecular weight 89 000–98 000) were purchased from Sigma-Aldrich (St. Louis, MO). Phosphate-buffered saline (PBS), fetal bovine serum (FBS), penicillin streptomycin, and (0.25% w/v) trypsin in 1 mM EDTA were obtained from Invitrogen (Carlsbad, CA). The RPMI-1640 culture medium was purchased from Gibco (Grand Island, NY). 3-[4,5Dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide (PI), phenylmethanesulfonyl fluoride (PMSF), and protease inhibitor cocktail were obtained from Molecular Probes (Carlsbad, CA). Primary antibodies against cyclin B1, b-actin, GAPDH, and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA).
Figure 1. Chemical structure of EVO.
were determined by a Malvern Zetasizer Nano ZS system (Malvern Instruments, Worcestershire, UK). The analyses were carried out at 25 C. The analysis of a sample consisted of three measurements was then taken an average value. The morphology of EVO-PLGA NPs analysis EVO-PLGA NPs morphology was characterized by a transmission electron microscopy (JEOL JEM-2100F, Tokyo, Japan). To examine NPs produced with the method of the phosphotungstic acid negative dyeing, in brief, a drop of EVO-PLGA NPs suspension in a 2% phosphotungstic acid solution (pH 7.2) was transferred to a copper grid for 2 min at room temperature for taking photographs.
HPLC analysis of EVO NPs
Differential scanning calorimetry study
The HPLC analysis of EVO was performed by a Waters system (Waters Corporation, Milford, MA) equipped with a UV detector and a Waters Symmetry C18 column (250 4.6 mm; 5 mm). The injection volume was 10 mL and the temperature of the column oven was set at 30 C. The mobile phase comprised of methanol and water containing 0.5% triethylamine in water (methanol:water, 60:40, v/v). The flow rate was 1.0 mL/min at a wavelength of 225 nm. All samples were analyzed for three times.
The crystal transformation of the NPs system was characterized by a differential scanning calorimetry (DSC-60 Calorimeter; Shimadzu Corporation, Kyoto, Japan). Five milligrams of each prepared samples of EVO, PLGA, physical mixture (the ratio of EVO and PLGA was 1:10), and EVOPLGA NPs were sealed in an aluminum crucible and heated at a rate of 10 C/min from 35 to 300 C under a nitrogen atmosphere.
Preparation of EVO-PLGA NPs
The entrapment efficiency of EVO-PLGA NPs was measured by a Waters system (Waters Corporation, Milford, MA) after filtration and appropriate dilution with acetonitrile and drug content in the NPs was determined. The entrapment efficiency of the nanoparticles was accurately calculated with the actual initial weight of the drug and the weight of free drug in the aqueous phase after filtration of suspension. Encapsulation efficiency and drug-loading content were calculated as follows:
EVO-PLGA NPs were prepared by solvent evaporation technique (Figure 1). In brief, PLGA (100 mg) and EVO (10 mg) were dissolved in acetone to gain organic phase. Then, organic solution was added dropwise to 1.5% (w/v) PVA solution to obtain the aqueous phase. The emulsion was sonicated for 5 min at 60 W and then gently stirred at room temperature for 12 h to eliminate the organic phase. The nanoparticles were centrifuged at 15 000 rpm for 15 min and then washed thrice with distilled water, followed by freeze drying to get the dry powder overnight. The EVO-PLGA NPs were stored at 4 C in refrigerator until use. Characterization of EVO-PLGA NPs Particle size, size distribution, and zeta potential The solution of EVO-PLGA NPs was prepared in distilled water to avoid multi-scattering phenomena. The mean particle size, polydispersity index (PDI) and zeta potential of NPs
Drug entrapment efficiency and drug-loading content
Encapsulation efficiencyð%Þ Theoretical drug content actual drug Free drug 100% ¼ Theoretical drug content Drug loading contentð%Þ Theoretical drug content actual drug Entrapment efficiency 100% ¼ Theoretical drug content þTheoretical PLGA content
EVO-PLGA nanoparticles
DOI: 10.3109/10717544.2014.920936
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In vitro release profile To study the EVO release rate, in vitro release of EVO from physical mixture and EVO-PLGA NPs was performed simulating physiologic conditions (37 C, PBS buffer, pH 7.4) as medium by the dialysis bag method. The dialysis bag (12 000–14 000 molecular weight cut-off) could retain NPs but allow the free EVO diffused into dissolution medium. The dialysis bag was put in conical flasks and then 5 mL PBS was put in, respectively. The conical flasks were kept in a shaker at 37 C at 100 rpm. The profiles were performed over 7 days. At the set time, a volume of 1 mL release medium was taken out from and then immediately followed by adding the equal volume of PBS. Furthermore, the in vitro release experiments of EVO from physical mixture and EVO-PLGA NPs were measured by a reversed-phase HPLC method. The release profiles were individually performed in triplicate and then calculated as average value. Cell culture Human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (Manassas, VA), and cultured in RPMI-1640 supplemented with 10% FBS and 100 U/mL penicillin and 100 mg/mL streptomycin antibiotics at 37 C in a humidified atmosphere containing 5% CO2. The medium was changed every other day. MTT assay MCF-7 cells were seeded at a density of 2.5 103 cell/ 100 mL/well in 96-well plates. When reached 60% confluency, the cells were treated with different concentrations (0, 0.1, 0.2, 0.5, 1, 2, 4, 8, and 16 mM) of EVO and equivalent doses of EVO-PLGA NPs for 48 h. Cells were incubated with MTT reagent (0.5 mg/mL) for 4 h at 37 C. The medium was discarded, and then 100 mL DMSO was added to dissolve the production of formazan crystals. The final solution was determined by a multilabel counter at the wavelength of 570 nm (Wallac VICTOR3Ô; Perkin Elmer, Waltham, MA). Observation of cell morphologic changes MCF-7 human breast cancer cells were seeded into 6-well plates and treated with different concentrations of EVO, PLGA, and EVO-PLGA NPs for 24 h, respectively. The cellular morphology was observed with an AxioCam HRc CCD camera (Oberkochen, Germany). Colony formation assay When a single cell proliferate in vitro over six generations, it will form the cell colony containing 450 cells and the size is between 0.3 and 1.0 mm. Cell colony formation rate is dependent on the ability of a single cell to survive. MCF-7 cells were seeded into 6-well plates at a density of 200 cells per well. After 72 h, cells were treated with EVO (0.5, 1 mM), EVO-PLGA NPs (0.5, 1 mM, the equivalent amount of EVO), and PLGA (the equivalent amount of PLGA in EVO-PLGA NPs). After 10 days, cells were fixed with 4% paraformaldehyde, and then stained with crystal violet staining solution (Beyotime Institute of Biotechnology, Shanghai, China).
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The typical images of the cells were photographed by a common CANON camera. Cell cycle analysis MCF-7 cells were seeded into 6-well plates. When cells reached 60% confluency, the cells were treated with EVO (0.5, 1 mM), EVO-PLGA NPs (0.5, 1 mM, the equivalent amount of EVO), and PLGA (the equivalent amount of PLGA in EVO-PLGA NPs). After being washed twice with PBS, cells were harvested and collected by centrifugation at 350 g for 5 min, and then were fixed with 70% ethanol and then were stored at 20 C overnight. Next, cells were collected by centrifugation and stained by 100 mL PI staining solution (5 mg/mL RNase and 20 mg/mL PI) at room temperature for 30 min avoiding light, followed by analysis with a flow cytometer (Becton Dickinson FACS CantoÔ, Franklin Lakes, NJ). More than 10 000 events were counted for each sample. The percentages of cell distributions in G0/G1, G2/M, and S-phases were analyzed by software FlowJo version 7.6.1 (Tree Star, Inc., Ashland, OR). Western blotting To determine the effect of EVO, PLGA and EVO-PLGA NPs on cyclin B1 and b-actin, the MCF-7 cells were treated with EVO (0.5, 1 mM), EVO-PLGA NPs (0.5, 1 mM, the equivalent amount of EVO), and PLGA (the equivalent amount of PLGA in EVO-PLGA NPs) for 24 h. After being treated with drugs, cells were washed twice with PBS and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, China) containing 1% PMSF and 1% protease inhibitor cocktail (Pierce, Rockford, IL). The concentrations of protein were determined with BCA protein assay kit (Pierce, Rockford, IL). Equivalent amounts of protein samples from each group were separated by electrophoresis using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), followed by transferring onto polyvinylidene difluoride (PVDF) membrane. After being blocked in 5% non-fat dried milk with PBS with Tween-20 (PBST) buffer for 1 h, the membrane was incubated with various primary antibodies (1:1000) at 4 C overnight. GAPDH (1:2000) was used as loading control. PVDF membranes were washed thrice in PBST buffer, and then were incubated with 1:5000 dilutions of the corresponding second antibodies. Specific protein bands were visualized using an ECL advanced Western blotting detection kit (GE Healthcare, Amersham, UK). The density of the bands was quantified by Quantity One Software (Bio-Rad, Hercules, CA). Statistical analysis Data were presented as the mean ± standard deviation. The data were submitted to one-way analysis of variance and Tukey’s multiple comparison test using the GraphPad Prism 5.0 software program (San Diego, CA). A value of p 50.05 was considered to be statistically significant for all the parameters evaluated.
Results and discussion Preparation of EVO-PLGA NPs EVO-PLGA NPs were prepared by a simple emulsification/ solvent evaporation method using PVA as stabilizer as
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Figure 2. (A) Solubility of EVO (physical mixture EVO and EVO-PLGA NPs) in PBS (pH ¼ 7.4); EVO (5 mg) was insoluble in PBS (pH 7.4), while EVO-PLGA NPs were fully soluble in aqueous solution; (B) TEM images of EVO-PLGA NPs; (C) and (D) particle size distribution and zeta potential of EVO-PLGA NPs.
previously reported (Li et al., 2008; Khalil et al., 2013). In screening the best prescription, it was found that the loading efficiency of EVO-PLGA NPs was strongly linked to external conditions. The ratios of drug and PLGA, organic phase and aqueous phase, as well as the optimal stirring time, stirring speed, ultrasonic power, and time were all investigated. Finally, we found that the optimal preparation condition was prepared as below, the ratio of PLGA and drug was 10:1; the ratio of organic phase and aqueous phase was 1:5; stirring gently for 12 h; sonicating 5 min at 60 W. As shown in Figure 2(A), the physical mixture of PLGA and EVO was insoluble in PBS (pH 7.4), while EVO-PLGA NPs were fully dissolved in aqueous solution. As PLGA was made of two parts, hydrophilic part was lactic acid and hydrophobic part was glycolic acid, the hydrophilic character of glycolic acid kept NPs suspended in aqueous phase, while the hydrophobic core of lactic acid could wrap the hydrophobic drugs. All in all, the obtained EVO-PLGA NPs showed good loading capacity in encapsulating EVO. Characterization of EVO-PLGA NPs PLGA with different molecular weight were used to prepared nanoparticles. As shown in Table 1, the drug-loading content and the particle sizes closely correlated with the molecular weight of PLGA; the larger the molecular weight was,
Table 1. Influence of copolymer composition on nanoparticle properties.
Copolymers
Loading content Encapsulation (%) efficiency (%)
4.5 ± 0.1 PLGA(50:50,10 000) PLGA(50:50,30 000–60 000) 5.8 ± 0.3 PLGA(75:25,76 000–115 000) 5.9 ± 0.1
50.1 ± 1.4 63.8 ± 1.7 65.1 ± 0.2
Particle size (nm)
PDI
151.24 ± 0.6 0.262 ± 0.01 157.36 ± 1.7 0.273 ± 0.01 283.34 ± 2.1 0.319 ± 0.06
the higher the drug-loading content and larger the particle size was. In order to achieve a higher drug-loading content and a smaller particle size, the molecular weight of PLGA(50:50,30 000–60 000) was best chosen for the further study. The particle size and zeta potential were important characteristics of NPs. The particle size should be small enough (5200 nm) to evade detection and destruction by the reticuloendothelial system so as to achieve prolonged circulation time and passive targeting to cancers via the EPR effect. The absolute value of zeta potential should be high enough to ensure a high stability against coalescence. As shown in Figure 2(C and D), the mean particle size and zeta potential of the EVO-PLGA NPs was 157.4 ± 1.7 nm, with a monomodal nanoparticle size distribution and low PDI. And the zeta potential was 23.6 ± 1.3 mV, indicating that the system was physically stable.
EVO-PLGA nanoparticles
DOI: 10.3109/10717544.2014.920936
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Table 2. Characteristic of EVO-PLGA NPs. Preparations
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EVO-PLGA NPs PLGA NPs
Particle size (nm)
PDI
Zeta potential (mV)
Encapsulation efficiency (%)
Loading content (%)
157.36 ± 1.7 128.81 ± 0.3
0.273 ± 0.01 0.265 ± 0.03
23.6 ± 1.3 25.3 ± 0.07
63.8 ± 1.7 –
5.8 ± 0.3 –
Figure 3. The DSC curves of physical mixture EVO, PLGA, and EVO-PLGA NPs.
Figure 4. In vitro cumulative percent release of EVO-loaded PLGA nanoparticles and EVO physical mixture in phosphate buffer at 37 C.
The morphology of EVO-PLGA NPs analysis
In vitro release profile
Transmission electron microscopy (TEM) image of EVOPLGA NPs was employed and micrographs are presented in Figure 2(B). It was shown that spherical nanoparticles had smooth surfaces and particle size 100 nm. The analysis by dynamic light scattering also proved that the nanoparticles had a monomodal nanoparticle size distribution and low PDI as shown in Figure 2(C) and Table 2.
The in vitro release behavior of EVO from physical mixture and EVO-PLGA NPs were studied for more than 180 h as shown in Figure 4. As it was shown, drug release from physical mixture and EVO-PLGA NPs occurred in a biphasic manner of burst release and sustained release. Due to the burst effect, the drug of physical mixture was released fast and finished in 20 h. It was found that there was an initial burst release according to the accumulative release rate of EVO from EVO-PLGA NPs, and this result may be due to EVO adhered on the surface of NPs was separated from PLGA into the dissolution medium. A sustained EVO release to 70 % was possessed for EVOPLGA NPs over 180 h. Thus, the polymer PLGA could prevent EVO from burst release and could control the release rate of EVO. Additionally, in vitro release curve suggest that EVO-PLGA NPs possessed a remarkable time prolongation effect on EVO release and superior physical long-time stability.
DSC study The DSC curves of PLGA, EVO, physical mixture (the ratio of EVO and PLGA was 1:10) and EVO-PLGA NPs are shown in Figure 3. It was possible that an interaction between EVO and PLGA existed after comparing these corresponding DSC curves. EVO exhibited a sharp peak because the thermal decomposition. PLGA had no distinct melting point because it was amorphous in nature (Mukerjee & Vishwanatha, 2009). Meanwhile, the characteristic peaks of EVO and PLGA could be found in physical mixture. However, we could observe from the curve of EVO-PLGA NPs that there was not only new characteristic peak but also the sharp peak of EVO disappeared, evidencing that most EVO in NPs were in an amorphous and a new effect between EVO and PLGA has occurred to change their thermal performance. We could conclude that EVO had been encapsulated in PLGA NPs. DSC was successfully used to characterize the nanoparticle systems of EVO-PLGA NPs. Drug entrapment efficiency and drug-loading content Encapsulation efficiency directly affects the efficiency of administration and treatment. The entrapment efficiency and drug-loading content of EVO-PLGA NPs were 63.8 ± 1.7 and 5.8 ± 0.3% (Table 2).
EVO-PLGA NPs enhanced the toxicity of EVO to MCF-7 cells MTT assay was used to evaluate the therapeutic potential of the formulation. Groups of untreated cells were served as controls. As shown in Figure 5, in contrast to the blank PLGA nanoparticles, 0.5 mM of EVO-PLGA NPs efficiently triggered inhibition of cancer cell proliferation after 48 h treatment. The cell proliferation was decreased by 450% after 48-h incubation with 16 mM of EVO-PLGA NPs, and less than 50% with 16 mM of EVO. The results demonstrated that EVO-PLGA NPs were more effective in arresting cell growth as compared to EVO at the concentrations of 2–16 mM.
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Cell morphologic changes
Colony formation assay
EVO treatment induced significant morphological changes in MCF-7 breast cancer cells. The cells were seeded in a 6-well plate, and incubated with EVO or EVO-PLGA NPs for 24 h. The 6-well plate was observed under inverted microscope. After incubating with 0.5 mM EVO or EVO-PLGA NPs, the long spindle-shaped cells became round and the intercellular gaps were found to be widened. The refraction of cells increased in the treated groups (Figure 6).
Cell colony formation rate is dependent on the ability of a single cell to survive. As shown in Figure 7, no visible colonies containing more than 50 cells were observed after 0.5 mM EVO or EVO-PLGA NPs treatment. It was indicated that both EVO and EVO-PLGA NPs inhibited the growth and division of breast cancer MCF-7 cells, and decreased the amount of clones at low doses. Cell cycle analysis Previous studies had been demonstrated that EVO inhibits the proliferation of cancer cells by blocking cell cycle progression (Huang et al., 2004, 2005; Rasul et al., 2012; Du et al., 2013). An analysis of DNA content in the samples treated with PLGA and EVO-PLGA NPs has been respectively performed to test whether the reduced proliferation observed was due to cell cycle arrest. Results showed that PLGA treatment did not affect the cell cycle phases in comparison with control group. In contrast, a significant G2/M block was observed in samples treated with 0.5 mM EVO (Figure 8). Moreover, a similar change in cell cycle was presented in samples treated with 1 mM EVO-PLGA NPs. These results suggested that the cytotoxic activity of EVO-PLGA NPs was essentially attributed to the cell cycle arrest induced by the release of EVO.
Figure 5. Cell proliferation was assessed after treating cells with EVO and EVO-loaded PLGA nanoparticles in MCF-7 cells by MTT assay. Cell viability was expressed as a percentage of the control (untreated cells). All assays were performed in six replicates and repeated at least three times (n 3).
EVO-PLGA NPs altered the expression of cell cycle-related proteins In order to examine the effect of the molecular events involved in EVO activity on cell cycle progression, we studied
Figure 6. Morphologic changes of MCF-7 cells. (A) ctrl, (B) 0.5 mM EVO, (C) 1 mM EVO, (D) PLGA (the equivalent amount of PLGA in 1 mM EVOPLGA NPs), (E) 0.5 mM EVO-PLGA NPs, (F) 1 mM EVO-PLGA NPs. Microscope observation showed that EVO and EVO-PLGA NPs treatment induced morphologic changes in MCF-7 cells.
DOI: 10.3109/10717544.2014.920936
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Figure 7. Colony formation assay indicated that treatment of EVO and EVO-PLGA NPs made a significant effect for the proliferation of MCF-7 cells.
Figure 8. Cell cycle analysis. Cells were treated for 24 h with different concentrations of EVO or EVO-PLGA NPs. Cells were fixed with ethanol, stained with PI (50 mg/mL) and analyzed by flow cytometry (Becton Dickinson FACS CantoÔ, Franklin Lakes, NJ). At least 10 000 events were counted for each sample. The percentages of cell distribution in G0/G1, G2/M, and S-phases were analyzed by Modfit 3.0 software (Topsham, ME). Relative distribution of cell population in the cell cycle phases is presented. (A) ctrl, (B) 0.5 mM EVO, (C) 1 mM EVO, (D) PLGA (the equivalent amount of PLGA in 1 mM EVO-PLGA NPs), (E) 0.5 mM EVO-PLGA NPs, the equivalent amount of 0.5 mM EVO, and (F) 1 mM EVO-PLGA NPs, the equivalent amount of 1 mM EVO.
the effect of the EVO on the expression of cyclin B1 as a protein that pivotal for G2/M transition. G2/M transition requires activity of cyclin-dependent kinase, which is regulated by cyclin B1 (Kimura et al., 1998). Figure 9 represents a typical Western blot result. An increase in the level of cyclin B1 was observed after 24 h treatment with 0.5 mM EVO or EVO-PLGA NPs. Cyclin B1 is a regulatory protein involved in mitosis. These results suggested that changes in the expression of G2/M transition regulating proteins might contribute to the EVO-mediated cell cycle arrest of MCF-7 breast cancer cells.
Actin filaments are major constituent of cytoskeleton (Chazotte, 2010) and highly conserved proteins, which were used as an internal control in western blot analysis and PCR. Meanwhile, it was demonstrated that b-actin specifically controls cell growth, cell cycle, migration and gene expression through controlling the cellular G-actin pool (Bunnell et al., 2011). Furthermore, it possesses a number of toxins that interfere with actin dynamics. Cytocalasin D, an alkaloid produced by fungi, is a potent inhibitor of actin polymerization by binding to the (+) end of F-actin and inhibiting the addition of new monomers (Cooper, 1987). Moreover,
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Figure 9. Western blotting analysis. The proteins of cell extracts from treated and untreated cells were prepared as previous mentioned, and fractionated by SDS–PAGE. Primary antibodies including cyclin B1 and b-actin were used. Rabbit monoclonal antibody against GAPDH is used as an internal control to normalize the quantity of protein loaded. The data presented the density ratios of each protein to GADPH.
disruption of actin dynamics activates the p53-dependent pathways causing arrest of cell cycle (Rubtsova et al., 1998). We hypothesize that the disruption of actin microfilaments by EVO may attribute to its anti-proliferation effect on MCF-7 cells, which remains to be investigated by further studies.
Conclusions In this study, a nanoparticulate delivery system based on PLGA nanoparticles (EVO-PLGA NPs) of EVO on breast cancer cells was prepared, accompanied with related physical and chemical characterization, as well as in vitro anticancer evaluation. The obtained EVO-PLGA NPs could persistently control the release of EVO for 4180 h. Furthermore, MTT assessment and colony formation assay showed that EVOPLGA NPs could enhance the toxicity and the proliferation inhibition effect of EVO against MCF-7 breast cancer cells. Moreover, EVO-PLGA NPs did not strengthen the G2/M blocking effect of EVO at 24-h incubation, suggesting that the nanoparticulate formulation of EVO could sustain the release of EVO in breast cancer cells. Therefore, it is concluded that PLGA nanoparticles are capable of delivering EVO over a prolonged period, which make it a potential candidate for cancer therapy.
Declaration of interest The authors declare no conflicts of interest in this work. This study was supported by the Macao Science and Technology Development Fund (077/2011/A3, 102/2012/A3), the Research Fund of the University of Macau (MYRG 107 (Y1-Y3)-ICMS13-HCW, MYRG 208 (Y3-L4)-ICMS11WYT, MRG012/WYT/2013/ICMS), and the State Key Laboratory of Natural and Biomimetic Drugs (K20130213).
References Acharya S, Sahoo SK. (2011). PLGA nanoparticles containing various anticancer agents and tumour delivery by EPR effect. Adv Drug Deliv Rev 63:170–83.
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Anderson JM, Shive MS. (1997). Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev 28:5–24. Beral V, Banks E, Reeves G, et al. (2003). Breast cancer and hormonereplacement therapy: the Million Women Study. Lancet 362:1330–1. Bunnell TM, Burbach BJ, Shimizu Y, Ervasti JM. (2011). b-Actin specifically controls cell growth, migration, and the G-actin pool. Mol Biol Cell 22:4047–58. Chazotte B. (2010). Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging. Cold Spring Harbour Protocol 2010:pdb.prot4947. Chen F, Li S, Li D, et al. (2012). Transdermal behaviors comparisons among Evodia rutaecarpa extracts with different purity of evodiamine and rutaecarpine and the effect of topical formulation in vivo. Fitoterapia 83:954–60. Chen MC, Yu CH, Wang SW, et al. (2010). Anti-proliferative effects of evodiamine on human thyroid cancer cell line ARO. J Cell Biochem 110:1495–503. Chung Y-I, Kim JC, Kim YH, et al. (2010). The effect of surface functionalization of PLGA nanoparticles by heparin-or chitosanconjugated Pluronic on tumor targeting. J Control Release 143: 374–82. Cooper JA. (1987). Effects of cytochalasin and phalloidin on actin. J Cell Biol 105:1473–8. Danhier F, Ansorena E, Silva JM, et al. (2012). PLGA-based nanoparticles: an overview of biomedical applications. J Control Release 161:505–22. Dong G, Wang S, Miao Z, et al. (2012). New tricks for an old natural product: discovery of highly potent evodiamine derivatives as novel antitumor agents by systemic structure–activity relationship analysis and biological evaluations. J Med Chem 55:7593–613. Du J, Wang XF, Zhou QM, et al. (2013). Evodiamine induces apoptosis and inhibits metastasis in MDAMB-231 human breast cancer cells in vitro and in vivo. Oncol Rep 30:685–94. Fei XF, Wang BX, Li TJ, et al. (2003). Evodiamine, a constituent of Evodiae Fructus, induces anti-proliferating effects in tumor cells. Cancer Sci 94:92–8. Huang DM, Guh JH, Huang YT, et al. (2005). Induction of mitotic arrest and apoptosis in human prostate cancer pc-3 cells by evodiamine. J Urol 173:256–61. Huang YC, Guh JH, Teng CM. (2004). Induction of mitotic arrest and apoptosis by evodiamine in human leukemic T-lymphocytes. Life Sci 75:35–49. Jiang J, Hu C. (2009). Evodiamine: a novel anti-cancer alkaloid from Evodia rutaecarpa. Molecules 14:1852–9. Khalil NM, do Nascimento TCF, Casa DM, et al. (2013). Pharmacokinetics of curcumin-loaded PLGA and PLGA-PEG blend nanoparticles after oral administration in rats. Colloids Surf B Biointerfaces 101:353–60. Kimura K, Hirano M, Kobayashi R, et al. (1998). Phosphorylation and activation of 13S condensin by Cdc2 in vitro. Science 282:487–90. Li M, Rouaud O, Poncelet D. (2008). Microencapsulation by solvent evaporation: state of the art for process engineering approaches. Int J Pharm 363:26–39. Li-Weber M. (2013). Targeting apoptosis pathways in cancer by Chinese medicine. Cancer Lett 332:304–12. Manchanda R, Fernandez-Fernandez A, Nagesetti A, et al. (2010). Preparation and characterization of a polymeric (PLGA) nanoparticulate drug delivery system with simultaneous incorporation of chemotherapeutic and thermo-optical agents. Colloids Surf B Biointerfaces 75:260–7. Mukerjee A, Vishwanatha JK. (2009). Formulation, characterization and evaluation of curcumin-loaded PLGA nanospheres for cancer therapy. Anticancer Res 29:3867–75. Rasul A, Yu B, Zhong L, et al. (2012). Cytotoxic effect of evodiamine in SGC-7901 human gastric adenocarcinoma cells via simultaneous induction of apoptosis and autophagy. Oncol Rep 27:1481–7. Rubtsova SN, Kondratov RV, Kopnin PB, et al. (1998). Disruption of actin microfilaments by cytochalasin D leads to activation of p53. FEBS Lett 430:353–7. Schwarz NA, Spillane M, La Bounty P, et al. (2013). Effects of capsaicin and evodiamine ingestion on energy expenditure and fat oxidation at rest and after moderately-intense exercise in young men. International Journal of Exercise Science: Conference Proceedings, Vol. 2, Article 11.
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Takada Y, Kobayashi Y, Aggarwal BB. (2005). Evodiamine abolishes constitutive and inducible NF-kB activation by inhibiting IkBa kinase activation, thereby suppressing NF-kB-regulated antiapoptotic and metastatic gene expression, up-regulating apoptosis, and inhibiting invasion. J Biol Chem 280:17203–12. Tan Q, Liu S, Chen X, et al. (2012). Design and evaluation of a novel evodiamine-phospholipid complex for improved oral bioavailability. AAPS PharmSciTech 13:534–47. Torchilin V. (2011). Tumor delivery of macromolecular drugs based on the EPR effect. Adv Drug Deliv Rev 63:131–5.
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Xu S, Peng J, Li Y, et al. (2012). Pharmacokinetic comparisons of rutaecarpine and evodiamine after oral administration of Wu-Chu-Yu extracts with different purities to rats. J Ethnopharmacol 139:395–400. Yang J, Cai X, Lu W, et al. (2012). Evodiamine inhibits STAT3 signaling by inducing phosphatase shatterproof 1 in hepatocellular carcinoma cells. Cancer Lett 328:243–51. Yu H, Jin H, Gong W, et al. (2013). Pharmacological actions of multitarget-directed evodiamine. Molecules 18:1826–43.
RESEARCH ARTICLE
Preparation, characterization, and anticancer efficacy of evodiamine-loaded PLGA nanoparticles Lidi Zou1*, Fengqian Chen1*, Jiaolin Bao1, Shengpeng Wang1, Lu Wang1, Meiwan Chen1,2, Chengwei He1, and Yitao Wang1 1
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China and Stake Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China
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Abstract
Keywords
Evodiamine (EVO) is a plant-derived indolequinazoline alkaloid with potential anticancer activity. However, low bioavailability caused by its poor water solubility limits it anticancer efficacy in clinic. To enhance the solubility and improve the bioavailability of EVO, a delivery system based on poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with EVO (EVO-PLGA NPs) for treating breast cancer was prepared in this study. The physicochemical characterization and in vitro antitumor evaluation of EVO-PLGA NPs were determined. EVO-PLGA NPs could persistently control the release of EVO for 180 h. 3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) assessment and colony formation assay showed that EVO-PLGA NPs could enhance the toxicity and the proliferation inhibition effect of EVO on MCF-7 breast cancer cells. EVO-PLGA NPs did not strengthen G2/M arrest effect of EVO-treated cells after 24h incubation. Meanwhile, EVO-PLGA NPs could increase the expression of cyclin B1 and decrease the expression of b-actin. Taken together, these results suggested that -PLGA NPs is promising for improving anticancer efficacy of EVO in breast cancer therapy.
Anticancer activity, breast cancer, evodiamine, formulation, PLGA nanoparticles
Introduction Breast cancer is one of the most prevalent malignancies in women diseases (Beral et al., 2003). However, numerous anticancer drugs have high toxicity, which leads to side effects, and have poor solubility that could decrease the drug efficacy. One approach to overcome these limitations is to develop a reliable nanoparticles (NPs) delivery strategies, which possesses several unique advantages, such as largely increased the aqueous solubility and improved bioavailability of the anticancer drugs, elongated the circulation time of blood, enhanced permeability and retention (EPR) so as to enhance the accumulation in the tumor sites and reduced systemic side effects (Torchilin, 2011). Thus, it is important to develop NPs delivery systems for anticancer agents. Evodiamine (EVO), an indolequinazoline alkaloid, is widely found in many medicinal plants of the tetradium family (Dong et al., 2012; Schwarz et al., 2013). It has been demonstrated that EVO possessed a plenty of pharmacologic activities, such as anti-obesity, anti-inflammatory (Chen et al., 2012), and anti-infectious effect (Li-Weber, 2013; Yu et al., 2013). Furthermore, many studies *These authors contributed equally to this work. Address for correspondence: Dr Meiwan Chen and Dr Chengwei He, State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Av. Padre Tomas Pereira S.J., Taipa, Macau, China. E-mail: [email protected] (M. Chen) and [email protected] (C. He)
History Received 14 March 2014 Revised 30 April 2014 Accepted 30 April 2014
declared that EVO was a promising anti-tumor agent (Jiang & Hu, 2009), which could suppress proliferation (Chen et al., 2010), induce apoptosis (Rasul et al., 2012), and inhibit metastasis (Takada et al., 2005) of cancer cells. These capabilities were believed to function well through the effects of EVO, which could alter the balance of Bcl2 and Bax gene expression by the caspase pathway in cancer cells (Fei et al., 2003). Furthermore, EVO could not only inhibit signal transducer and activator of transcription-3 (STAT3) DNAbinding activity, but also down-regulate the expression of STAT3-mediated genes leading to the suppression of proliferation and induction of cell apoptosis and cell cycle arrest (Yang et al., 2012). EVO, one of dissociative alkaloid in nature, possesses poor solubility in aqueous solution so as to limit EVO in systemic bioavailability and clinical efficacy (Tan et al., 2012; Xu et al., 2012). To increase the solubility of EVO, a NPs delivery system based on poly (lactic-co-glycolic acid) (PLGA) were prepared by a single emulsion (o/w) solvent evaporation technique (Manchanda et al., 2010). PLGA, which is approved by European Medicine Agency as non-toxic materials to prepare nanoparticle drug delivery systems (Danhier et al., 2012), was chosen to be the delivery material in this study for its well biodegradability property (Acharya & Sahoo, 2011; Anderson & Shive, 1997) and its ability of controlling sustained release (Chung et al., 2010). The EVO-loaded PLGA NPs (EVO-PLGA NPs) were developed to investigate the proliferation inhibition effect of breast cancer MCF-7 cells, aiming
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to disclose the relevant factors related to the pharmacologic activity of EVO-loaded PLGA NPs. EVO-PLGA NPs were characterized by its encapsulation efficiency, particle size, zeta potential, in vitro drug release behavior, and anticancer activity to MCF-7 human breast adenocarcinoma cell line. These results suggested that the application of EVO-PLGA NPs delivery system is promising for improvement of anticancer efficacy in cancer therapy.
Materials and methods
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Materials EVO was obtained from Chengdu Must Bio-Technology Co., Ltd, and the purity was determined to be 98% by highperformance liquid chromatography (HPLC). PLGA with different molecular weights and polyvinyl alcohol (PVA, molecular weight 89 000–98 000) were purchased from Sigma-Aldrich (St. Louis, MO). Phosphate-buffered saline (PBS), fetal bovine serum (FBS), penicillin streptomycin, and (0.25% w/v) trypsin in 1 mM EDTA were obtained from Invitrogen (Carlsbad, CA). The RPMI-1640 culture medium was purchased from Gibco (Grand Island, NY). 3-[4,5Dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide (PI), phenylmethanesulfonyl fluoride (PMSF), and protease inhibitor cocktail were obtained from Molecular Probes (Carlsbad, CA). Primary antibodies against cyclin B1, b-actin, GAPDH, and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA).
Figure 1. Chemical structure of EVO.
were determined by a Malvern Zetasizer Nano ZS system (Malvern Instruments, Worcestershire, UK). The analyses were carried out at 25 C. The analysis of a sample consisted of three measurements was then taken an average value. The morphology of EVO-PLGA NPs analysis EVO-PLGA NPs morphology was characterized by a transmission electron microscopy (JEOL JEM-2100F, Tokyo, Japan). To examine NPs produced with the method of the phosphotungstic acid negative dyeing, in brief, a drop of EVO-PLGA NPs suspension in a 2% phosphotungstic acid solution (pH 7.2) was transferred to a copper grid for 2 min at room temperature for taking photographs.
HPLC analysis of EVO NPs
Differential scanning calorimetry study
The HPLC analysis of EVO was performed by a Waters system (Waters Corporation, Milford, MA) equipped with a UV detector and a Waters Symmetry C18 column (250 4.6 mm; 5 mm). The injection volume was 10 mL and the temperature of the column oven was set at 30 C. The mobile phase comprised of methanol and water containing 0.5% triethylamine in water (methanol:water, 60:40, v/v). The flow rate was 1.0 mL/min at a wavelength of 225 nm. All samples were analyzed for three times.
The crystal transformation of the NPs system was characterized by a differential scanning calorimetry (DSC-60 Calorimeter; Shimadzu Corporation, Kyoto, Japan). Five milligrams of each prepared samples of EVO, PLGA, physical mixture (the ratio of EVO and PLGA was 1:10), and EVOPLGA NPs were sealed in an aluminum crucible and heated at a rate of 10 C/min from 35 to 300 C under a nitrogen atmosphere.
Preparation of EVO-PLGA NPs
The entrapment efficiency of EVO-PLGA NPs was measured by a Waters system (Waters Corporation, Milford, MA) after filtration and appropriate dilution with acetonitrile and drug content in the NPs was determined. The entrapment efficiency of the nanoparticles was accurately calculated with the actual initial weight of the drug and the weight of free drug in the aqueous phase after filtration of suspension. Encapsulation efficiency and drug-loading content were calculated as follows:
EVO-PLGA NPs were prepared by solvent evaporation technique (Figure 1). In brief, PLGA (100 mg) and EVO (10 mg) were dissolved in acetone to gain organic phase. Then, organic solution was added dropwise to 1.5% (w/v) PVA solution to obtain the aqueous phase. The emulsion was sonicated for 5 min at 60 W and then gently stirred at room temperature for 12 h to eliminate the organic phase. The nanoparticles were centrifuged at 15 000 rpm for 15 min and then washed thrice with distilled water, followed by freeze drying to get the dry powder overnight. The EVO-PLGA NPs were stored at 4 C in refrigerator until use. Characterization of EVO-PLGA NPs Particle size, size distribution, and zeta potential The solution of EVO-PLGA NPs was prepared in distilled water to avoid multi-scattering phenomena. The mean particle size, polydispersity index (PDI) and zeta potential of NPs
Drug entrapment efficiency and drug-loading content
Encapsulation efficiencyð%Þ Theoretical drug content actual drug Free drug 100% ¼ Theoretical drug content Drug loading contentð%Þ Theoretical drug content actual drug Entrapment efficiency 100% ¼ Theoretical drug content þTheoretical PLGA content
EVO-PLGA nanoparticles
DOI: 10.3109/10717544.2014.920936
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In vitro release profile To study the EVO release rate, in vitro release of EVO from physical mixture and EVO-PLGA NPs was performed simulating physiologic conditions (37 C, PBS buffer, pH 7.4) as medium by the dialysis bag method. The dialysis bag (12 000–14 000 molecular weight cut-off) could retain NPs but allow the free EVO diffused into dissolution medium. The dialysis bag was put in conical flasks and then 5 mL PBS was put in, respectively. The conical flasks were kept in a shaker at 37 C at 100 rpm. The profiles were performed over 7 days. At the set time, a volume of 1 mL release medium was taken out from and then immediately followed by adding the equal volume of PBS. Furthermore, the in vitro release experiments of EVO from physical mixture and EVO-PLGA NPs were measured by a reversed-phase HPLC method. The release profiles were individually performed in triplicate and then calculated as average value. Cell culture Human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (Manassas, VA), and cultured in RPMI-1640 supplemented with 10% FBS and 100 U/mL penicillin and 100 mg/mL streptomycin antibiotics at 37 C in a humidified atmosphere containing 5% CO2. The medium was changed every other day. MTT assay MCF-7 cells were seeded at a density of 2.5 103 cell/ 100 mL/well in 96-well plates. When reached 60% confluency, the cells were treated with different concentrations (0, 0.1, 0.2, 0.5, 1, 2, 4, 8, and 16 mM) of EVO and equivalent doses of EVO-PLGA NPs for 48 h. Cells were incubated with MTT reagent (0.5 mg/mL) for 4 h at 37 C. The medium was discarded, and then 100 mL DMSO was added to dissolve the production of formazan crystals. The final solution was determined by a multilabel counter at the wavelength of 570 nm (Wallac VICTOR3Ô; Perkin Elmer, Waltham, MA). Observation of cell morphologic changes MCF-7 human breast cancer cells were seeded into 6-well plates and treated with different concentrations of EVO, PLGA, and EVO-PLGA NPs for 24 h, respectively. The cellular morphology was observed with an AxioCam HRc CCD camera (Oberkochen, Germany). Colony formation assay When a single cell proliferate in vitro over six generations, it will form the cell colony containing 450 cells and the size is between 0.3 and 1.0 mm. Cell colony formation rate is dependent on the ability of a single cell to survive. MCF-7 cells were seeded into 6-well plates at a density of 200 cells per well. After 72 h, cells were treated with EVO (0.5, 1 mM), EVO-PLGA NPs (0.5, 1 mM, the equivalent amount of EVO), and PLGA (the equivalent amount of PLGA in EVO-PLGA NPs). After 10 days, cells were fixed with 4% paraformaldehyde, and then stained with crystal violet staining solution (Beyotime Institute of Biotechnology, Shanghai, China).
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The typical images of the cells were photographed by a common CANON camera. Cell cycle analysis MCF-7 cells were seeded into 6-well plates. When cells reached 60% confluency, the cells were treated with EVO (0.5, 1 mM), EVO-PLGA NPs (0.5, 1 mM, the equivalent amount of EVO), and PLGA (the equivalent amount of PLGA in EVO-PLGA NPs). After being washed twice with PBS, cells were harvested and collected by centrifugation at 350 g for 5 min, and then were fixed with 70% ethanol and then were stored at 20 C overnight. Next, cells were collected by centrifugation and stained by 100 mL PI staining solution (5 mg/mL RNase and 20 mg/mL PI) at room temperature for 30 min avoiding light, followed by analysis with a flow cytometer (Becton Dickinson FACS CantoÔ, Franklin Lakes, NJ). More than 10 000 events were counted for each sample. The percentages of cell distributions in G0/G1, G2/M, and S-phases were analyzed by software FlowJo version 7.6.1 (Tree Star, Inc., Ashland, OR). Western blotting To determine the effect of EVO, PLGA and EVO-PLGA NPs on cyclin B1 and b-actin, the MCF-7 cells were treated with EVO (0.5, 1 mM), EVO-PLGA NPs (0.5, 1 mM, the equivalent amount of EVO), and PLGA (the equivalent amount of PLGA in EVO-PLGA NPs) for 24 h. After being treated with drugs, cells were washed twice with PBS and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, China) containing 1% PMSF and 1% protease inhibitor cocktail (Pierce, Rockford, IL). The concentrations of protein were determined with BCA protein assay kit (Pierce, Rockford, IL). Equivalent amounts of protein samples from each group were separated by electrophoresis using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), followed by transferring onto polyvinylidene difluoride (PVDF) membrane. After being blocked in 5% non-fat dried milk with PBS with Tween-20 (PBST) buffer for 1 h, the membrane was incubated with various primary antibodies (1:1000) at 4 C overnight. GAPDH (1:2000) was used as loading control. PVDF membranes were washed thrice in PBST buffer, and then were incubated with 1:5000 dilutions of the corresponding second antibodies. Specific protein bands were visualized using an ECL advanced Western blotting detection kit (GE Healthcare, Amersham, UK). The density of the bands was quantified by Quantity One Software (Bio-Rad, Hercules, CA). Statistical analysis Data were presented as the mean ± standard deviation. The data were submitted to one-way analysis of variance and Tukey’s multiple comparison test using the GraphPad Prism 5.0 software program (San Diego, CA). A value of p 50.05 was considered to be statistically significant for all the parameters evaluated.
Results and discussion Preparation of EVO-PLGA NPs EVO-PLGA NPs were prepared by a simple emulsification/ solvent evaporation method using PVA as stabilizer as
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Figure 2. (A) Solubility of EVO (physical mixture EVO and EVO-PLGA NPs) in PBS (pH ¼ 7.4); EVO (5 mg) was insoluble in PBS (pH 7.4), while EVO-PLGA NPs were fully soluble in aqueous solution; (B) TEM images of EVO-PLGA NPs; (C) and (D) particle size distribution and zeta potential of EVO-PLGA NPs.
previously reported (Li et al., 2008; Khalil et al., 2013). In screening the best prescription, it was found that the loading efficiency of EVO-PLGA NPs was strongly linked to external conditions. The ratios of drug and PLGA, organic phase and aqueous phase, as well as the optimal stirring time, stirring speed, ultrasonic power, and time were all investigated. Finally, we found that the optimal preparation condition was prepared as below, the ratio of PLGA and drug was 10:1; the ratio of organic phase and aqueous phase was 1:5; stirring gently for 12 h; sonicating 5 min at 60 W. As shown in Figure 2(A), the physical mixture of PLGA and EVO was insoluble in PBS (pH 7.4), while EVO-PLGA NPs were fully dissolved in aqueous solution. As PLGA was made of two parts, hydrophilic part was lactic acid and hydrophobic part was glycolic acid, the hydrophilic character of glycolic acid kept NPs suspended in aqueous phase, while the hydrophobic core of lactic acid could wrap the hydrophobic drugs. All in all, the obtained EVO-PLGA NPs showed good loading capacity in encapsulating EVO. Characterization of EVO-PLGA NPs PLGA with different molecular weight were used to prepared nanoparticles. As shown in Table 1, the drug-loading content and the particle sizes closely correlated with the molecular weight of PLGA; the larger the molecular weight was,
Table 1. Influence of copolymer composition on nanoparticle properties.
Copolymers
Loading content Encapsulation (%) efficiency (%)
4.5 ± 0.1 PLGA(50:50,10 000) PLGA(50:50,30 000–60 000) 5.8 ± 0.3 PLGA(75:25,76 000–115 000) 5.9 ± 0.1
50.1 ± 1.4 63.8 ± 1.7 65.1 ± 0.2
Particle size (nm)
PDI
151.24 ± 0.6 0.262 ± 0.01 157.36 ± 1.7 0.273 ± 0.01 283.34 ± 2.1 0.319 ± 0.06
the higher the drug-loading content and larger the particle size was. In order to achieve a higher drug-loading content and a smaller particle size, the molecular weight of PLGA(50:50,30 000–60 000) was best chosen for the further study. The particle size and zeta potential were important characteristics of NPs. The particle size should be small enough (5200 nm) to evade detection and destruction by the reticuloendothelial system so as to achieve prolonged circulation time and passive targeting to cancers via the EPR effect. The absolute value of zeta potential should be high enough to ensure a high stability against coalescence. As shown in Figure 2(C and D), the mean particle size and zeta potential of the EVO-PLGA NPs was 157.4 ± 1.7 nm, with a monomodal nanoparticle size distribution and low PDI. And the zeta potential was 23.6 ± 1.3 mV, indicating that the system was physically stable.
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Table 2. Characteristic of EVO-PLGA NPs. Preparations
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EVO-PLGA NPs PLGA NPs
Particle size (nm)
PDI
Zeta potential (mV)
Encapsulation efficiency (%)
Loading content (%)
157.36 ± 1.7 128.81 ± 0.3
0.273 ± 0.01 0.265 ± 0.03
23.6 ± 1.3 25.3 ± 0.07
63.8 ± 1.7 –
5.8 ± 0.3 –
Figure 3. The DSC curves of physical mixture EVO, PLGA, and EVO-PLGA NPs.
Figure 4. In vitro cumulative percent release of EVO-loaded PLGA nanoparticles and EVO physical mixture in phosphate buffer at 37 C.
The morphology of EVO-PLGA NPs analysis
In vitro release profile
Transmission electron microscopy (TEM) image of EVOPLGA NPs was employed and micrographs are presented in Figure 2(B). It was shown that spherical nanoparticles had smooth surfaces and particle size 100 nm. The analysis by dynamic light scattering also proved that the nanoparticles had a monomodal nanoparticle size distribution and low PDI as shown in Figure 2(C) and Table 2.
The in vitro release behavior of EVO from physical mixture and EVO-PLGA NPs were studied for more than 180 h as shown in Figure 4. As it was shown, drug release from physical mixture and EVO-PLGA NPs occurred in a biphasic manner of burst release and sustained release. Due to the burst effect, the drug of physical mixture was released fast and finished in 20 h. It was found that there was an initial burst release according to the accumulative release rate of EVO from EVO-PLGA NPs, and this result may be due to EVO adhered on the surface of NPs was separated from PLGA into the dissolution medium. A sustained EVO release to 70 % was possessed for EVOPLGA NPs over 180 h. Thus, the polymer PLGA could prevent EVO from burst release and could control the release rate of EVO. Additionally, in vitro release curve suggest that EVO-PLGA NPs possessed a remarkable time prolongation effect on EVO release and superior physical long-time stability.
DSC study The DSC curves of PLGA, EVO, physical mixture (the ratio of EVO and PLGA was 1:10) and EVO-PLGA NPs are shown in Figure 3. It was possible that an interaction between EVO and PLGA existed after comparing these corresponding DSC curves. EVO exhibited a sharp peak because the thermal decomposition. PLGA had no distinct melting point because it was amorphous in nature (Mukerjee & Vishwanatha, 2009). Meanwhile, the characteristic peaks of EVO and PLGA could be found in physical mixture. However, we could observe from the curve of EVO-PLGA NPs that there was not only new characteristic peak but also the sharp peak of EVO disappeared, evidencing that most EVO in NPs were in an amorphous and a new effect between EVO and PLGA has occurred to change their thermal performance. We could conclude that EVO had been encapsulated in PLGA NPs. DSC was successfully used to characterize the nanoparticle systems of EVO-PLGA NPs. Drug entrapment efficiency and drug-loading content Encapsulation efficiency directly affects the efficiency of administration and treatment. The entrapment efficiency and drug-loading content of EVO-PLGA NPs were 63.8 ± 1.7 and 5.8 ± 0.3% (Table 2).
EVO-PLGA NPs enhanced the toxicity of EVO to MCF-7 cells MTT assay was used to evaluate the therapeutic potential of the formulation. Groups of untreated cells were served as controls. As shown in Figure 5, in contrast to the blank PLGA nanoparticles, 0.5 mM of EVO-PLGA NPs efficiently triggered inhibition of cancer cell proliferation after 48 h treatment. The cell proliferation was decreased by 450% after 48-h incubation with 16 mM of EVO-PLGA NPs, and less than 50% with 16 mM of EVO. The results demonstrated that EVO-PLGA NPs were more effective in arresting cell growth as compared to EVO at the concentrations of 2–16 mM.
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Cell morphologic changes
Colony formation assay
EVO treatment induced significant morphological changes in MCF-7 breast cancer cells. The cells were seeded in a 6-well plate, and incubated with EVO or EVO-PLGA NPs for 24 h. The 6-well plate was observed under inverted microscope. After incubating with 0.5 mM EVO or EVO-PLGA NPs, the long spindle-shaped cells became round and the intercellular gaps were found to be widened. The refraction of cells increased in the treated groups (Figure 6).
Cell colony formation rate is dependent on the ability of a single cell to survive. As shown in Figure 7, no visible colonies containing more than 50 cells were observed after 0.5 mM EVO or EVO-PLGA NPs treatment. It was indicated that both EVO and EVO-PLGA NPs inhibited the growth and division of breast cancer MCF-7 cells, and decreased the amount of clones at low doses. Cell cycle analysis Previous studies had been demonstrated that EVO inhibits the proliferation of cancer cells by blocking cell cycle progression (Huang et al., 2004, 2005; Rasul et al., 2012; Du et al., 2013). An analysis of DNA content in the samples treated with PLGA and EVO-PLGA NPs has been respectively performed to test whether the reduced proliferation observed was due to cell cycle arrest. Results showed that PLGA treatment did not affect the cell cycle phases in comparison with control group. In contrast, a significant G2/M block was observed in samples treated with 0.5 mM EVO (Figure 8). Moreover, a similar change in cell cycle was presented in samples treated with 1 mM EVO-PLGA NPs. These results suggested that the cytotoxic activity of EVO-PLGA NPs was essentially attributed to the cell cycle arrest induced by the release of EVO.
Figure 5. Cell proliferation was assessed after treating cells with EVO and EVO-loaded PLGA nanoparticles in MCF-7 cells by MTT assay. Cell viability was expressed as a percentage of the control (untreated cells). All assays were performed in six replicates and repeated at least three times (n 3).
EVO-PLGA NPs altered the expression of cell cycle-related proteins In order to examine the effect of the molecular events involved in EVO activity on cell cycle progression, we studied
Figure 6. Morphologic changes of MCF-7 cells. (A) ctrl, (B) 0.5 mM EVO, (C) 1 mM EVO, (D) PLGA (the equivalent amount of PLGA in 1 mM EVOPLGA NPs), (E) 0.5 mM EVO-PLGA NPs, (F) 1 mM EVO-PLGA NPs. Microscope observation showed that EVO and EVO-PLGA NPs treatment induced morphologic changes in MCF-7 cells.
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Figure 7. Colony formation assay indicated that treatment of EVO and EVO-PLGA NPs made a significant effect for the proliferation of MCF-7 cells.
Figure 8. Cell cycle analysis. Cells were treated for 24 h with different concentrations of EVO or EVO-PLGA NPs. Cells were fixed with ethanol, stained with PI (50 mg/mL) and analyzed by flow cytometry (Becton Dickinson FACS CantoÔ, Franklin Lakes, NJ). At least 10 000 events were counted for each sample. The percentages of cell distribution in G0/G1, G2/M, and S-phases were analyzed by Modfit 3.0 software (Topsham, ME). Relative distribution of cell population in the cell cycle phases is presented. (A) ctrl, (B) 0.5 mM EVO, (C) 1 mM EVO, (D) PLGA (the equivalent amount of PLGA in 1 mM EVO-PLGA NPs), (E) 0.5 mM EVO-PLGA NPs, the equivalent amount of 0.5 mM EVO, and (F) 1 mM EVO-PLGA NPs, the equivalent amount of 1 mM EVO.
the effect of the EVO on the expression of cyclin B1 as a protein that pivotal for G2/M transition. G2/M transition requires activity of cyclin-dependent kinase, which is regulated by cyclin B1 (Kimura et al., 1998). Figure 9 represents a typical Western blot result. An increase in the level of cyclin B1 was observed after 24 h treatment with 0.5 mM EVO or EVO-PLGA NPs. Cyclin B1 is a regulatory protein involved in mitosis. These results suggested that changes in the expression of G2/M transition regulating proteins might contribute to the EVO-mediated cell cycle arrest of MCF-7 breast cancer cells.
Actin filaments are major constituent of cytoskeleton (Chazotte, 2010) and highly conserved proteins, which were used as an internal control in western blot analysis and PCR. Meanwhile, it was demonstrated that b-actin specifically controls cell growth, cell cycle, migration and gene expression through controlling the cellular G-actin pool (Bunnell et al., 2011). Furthermore, it possesses a number of toxins that interfere with actin dynamics. Cytocalasin D, an alkaloid produced by fungi, is a potent inhibitor of actin polymerization by binding to the (+) end of F-actin and inhibiting the addition of new monomers (Cooper, 1987). Moreover,
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Figure 9. Western blotting analysis. The proteins of cell extracts from treated and untreated cells were prepared as previous mentioned, and fractionated by SDS–PAGE. Primary antibodies including cyclin B1 and b-actin were used. Rabbit monoclonal antibody against GAPDH is used as an internal control to normalize the quantity of protein loaded. The data presented the density ratios of each protein to GADPH.
disruption of actin dynamics activates the p53-dependent pathways causing arrest of cell cycle (Rubtsova et al., 1998). We hypothesize that the disruption of actin microfilaments by EVO may attribute to its anti-proliferation effect on MCF-7 cells, which remains to be investigated by further studies.
Conclusions In this study, a nanoparticulate delivery system based on PLGA nanoparticles (EVO-PLGA NPs) of EVO on breast cancer cells was prepared, accompanied with related physical and chemical characterization, as well as in vitro anticancer evaluation. The obtained EVO-PLGA NPs could persistently control the release of EVO for 4180 h. Furthermore, MTT assessment and colony formation assay showed that EVOPLGA NPs could enhance the toxicity and the proliferation inhibition effect of EVO against MCF-7 breast cancer cells. Moreover, EVO-PLGA NPs did not strengthen the G2/M blocking effect of EVO at 24-h incubation, suggesting that the nanoparticulate formulation of EVO could sustain the release of EVO in breast cancer cells. Therefore, it is concluded that PLGA nanoparticles are capable of delivering EVO over a prolonged period, which make it a potential candidate for cancer therapy.
Declaration of interest The authors declare no conflicts of interest in this work. This study was supported by the Macao Science and Technology Development Fund (077/2011/A3, 102/2012/A3), the Research Fund of the University of Macau (MYRG 107 (Y1-Y3)-ICMS13-HCW, MYRG 208 (Y3-L4)-ICMS11WYT, MRG012/WYT/2013/ICMS), and the State Key Laboratory of Natural and Biomimetic Drugs (K20130213).
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DOI: 10.3109/10717544.2014.920936
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