Materials Science and Engineering C 36 (2014) 7–13
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells Jin Liang a,b, Feng Li b, Yong Fang c, Wenjian Yang c, Xinxin An b, Liyan Zhao b, Zhihong Xin b, Lin Cao b, Qiuhui Hu b,c,⁎ a b c
Key Laboratory of Tea Biochemistry and Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, People's Republic of China College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People's Republic of China College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023, People's Republic of China
a r t i c l e
i n f o
Article history: Received 26 November 2012 Received in revised form 24 October 2013 Accepted 26 November 2013 Available online 5 December 2013 Keywords: Tea polyphenols Chitosan Nanoparticles Hepatoma Apoptosis
a b s t r a c t Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenolloaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs. © 2013 Published by Elsevier B.V.
1. Introduction Nowadays, cancer is one of the major dread diseases and chemoprevention is considered a valid approach to reduce the incidence of cancer [1]. Tea polyphenols (TP) from the dried leaves of the plant Camellia sinensis are known to possess strong antioxidant and anti-carcinogenic activities [2]. Although TP are widely used for the prevention and treatment of cancer, the therapeutic effects are limited due to their certain drawbacks, such as poor stability in gastrointestinal tract and limited bioavailability in vivo [3]. It has been reported that the biological activity of TP might depend on the form of their administration [4]. Therefore, a lot of research has been directed towards the development of effective methods to overcome the bottlenecks. Nanoparticles, especially those with highly controlled shapes, sizes and some unique physical and ⁎ Corresponding author at: College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People's Republic of China. Tel./fax: + 86 25 84399086. E-mail address: [email protected] (Q. Hu). 0928-4931/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.msec.2013.11.039
chemical properties including the antitumor activities [5], have been studied extensively as drug carriers for the improvement of the bioavailability of drug with poor absorption characteristics. Owing to their small size, prolonged circulation time, and sustained drug release profile, nano-sized polymeric nanoparticles bearing anticancer drugs have received increased attention for their ability to improve the efficacy of anticancer drugs [6]. Nanoparticles can facilitate controlled and targeted drug delivery of the encapsulated anticancer drugs with high efficacy and low side effects [7]. They not only have the ability to overcome biological barriers and accumulate preferentially in tumors, but also can recognize single cancer cells, which are useful to the detection and treatment of cancer. Chitosan, a deacetylated derivative of chitin, has been applied extensively as a functional biopolymer in food and pharmaceutical industry, and is well known for its abundant, renewable, non-toxic and biodegradable nature. Because of its unique chemical structure, chitosan has been investigated extensively in the development of controlled release drug delivery systems. Especially, the mucoadhesive property of chitosan can enhance drug transmucosal absorption and promote its
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J. Liang et al. / Materials Science and Engineering C 36 (2014) 7–13
sustained release [8,9]. It has been reported that chitosan nanoparticles as drug carriers with small particle size and positive surface charge can exhibit a higher antitumor activity [10]. Our previous studies showed that the tea polyphenol-loaded chitosan nanoparticles (TP-CNPs) could be obtained by ionic gelation using two biocompatible materials, namely N-carboxymethyl chitosan and chitosan hydrochloride, and the optimization of particle size and entrapment efficiency have been discussed [11]. However, the antitumor biological activity of TP-CNPs is still unknown. Therefore, in this study, the antitumor effects of the TP-CNP, TP and chitosan nanoparticles (CNPs) have been evaluated using HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, apoptosis detection, and cell cycle measurement, and the antitumor mechanism was also hypothesized. 2. Materials and methods
0.25 mg/mL) and TP-CNPs loaded with the same amount of TP. The untreated cells were used as the control. The plates were incubated in a humidified 5% CO2 balanced-air incubator at 37 °C for 48 h. Then 10 μL of 5 mg/mL MTT solution was added into each well and the plates were incubated for another 4 h, and then the medium was discarded. Dimethyl sulfoxide (100 μL) was added into each well of the 96-well plate, and the solution was vigorously mixed to dissolve tetrazolium dye. The absorbance of each well was measured by an enzyme-linked immunosorbent assay reader (BioTek; Austria) at a test wavelength of 570 nm and the cell cytotoxicity rate was calculated by the following equation: Cell cytotoxicity ¼ ½1–T=C 100% where C is the number of viable cells after 48 h of incubation without nanoparticles and T is the number of viable cells after 48 h of incubation with nanoparticles.
2.1. Materials 2.5. Morphological examination using microscope TP extract with a purity of 93% was obtained from dried green tea leaves by the solvent extraction method using 50% ethanol and then purified with H1020 resins (Nankai University Chemical Plant, Tianjin, China). HepG2 cells were obtained from Nanjing University (Nanjing, Jiangsu, China). All the other reagents used were of analytical grade.
100 μL HepG2 cells (1 × 105 cells/mL) were seeded on 96-well culture plates and TP-CNPs, CNPs and TP (1.0 mg/mL) were added into the cells, and after 48 h incubation, morphological changes of cells were examined under a fluorescent microscope (CKX41, Olympus Corporation, Tokyo, Japan).
2.2. The preparation of TP-CNPs 2.6. Transmission electron microscopy assay The TP-CNPs were prepared according to our previously reported optimized conditions [11,12]. Briefly, N-carboxymethyl chitosan (Mv = 61 kDa, Degree of deacetylation 83%) and chitosan hydrochloride (Mv = 90 kDa, Degree of deacetylation 85%) were dissolved in distilled water and the solutions of different concentrations were prepared by adding the extracts of TP into carboxymethyl chitosan solution. The optimal levels of carboxymethyl chitosan concentration, chitosan hydrochloride concentration and the amount of TP were 3.63 mg/mL, 1.19 mg/mL, and 10.94 mg, respectively. The main preparation process was the following steps. First, TP was added into the chitosan hydrochloride solution (30 mL), and then the carboxymethyl chitosan solution (12 mL) was added dropwise into the chitosan hydrochloride and TP mixed solution under stirring at room temperature, and continuously stirred for 30 min. The formation of nanoclusters was based on an ionic gelation interaction between positively-charged amine groups of chitosan hydrochloride chitosan and negatively-charged carboxyl groups of carboxymethyl chitosan. The nanocluster suspensions were immediately subjected to further analysis for their structure and size distribution characterization. The nanocluster powders as experimental samples were obtained by high speed centrifugation (12,000 rpm, 30 min) followed by freeze-drying process. Meanwhile, the CNPs without TP were prepared to use as the control group. 2.3. Cell culture Human hepatoma HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) equilibrated with 5% CO2 and 95% air at 37 °C. The medium was supplemented with 10% fetal calf serum (FCS), 50 mg/L streptomycin and 75 mg/L penicillin sulfate.
Changes in the ultrastructure of HepG2 cells after treatment with TPCNPs were observed using the transmission electron microscope (TEM) according to the previously published protocol [7]. Briefly, after incubation with TP-CNPs (1 mg/mL) at 37 °C for 24 h, HepG2 cells were collected by centrifugation and washed twice with PBS, and then fixated immediately in Karnovsky solution (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na cacodylate/HCl, pH 7.4). The postfixation was performed in 1% OsO4 with 1.5% potassium ferrocyanide, followed by dehydration and drying. After being trimmed, mounted and coated, the ultrathin sections were observed and photographed by using the TEM (JEOL H-7650, Hitachi High-Technologies Corporation, Tokyo, Japan). At the same time, the TEM images of TP and CNPs were also captured using the same method. 2.7. Determination of cell apoptosis The cell apoptosis was detected by annexin V/PI double stain assay. The annexin V/PI staining was performed as described previously [13]. Briefly, HepG2 cells (3 × 105 cells/well) were seeded in 24-well plates and incubated at 37 °C in a 5% CO2 incubator overnight. After treatment with tea polyphenols at 1.0 mg/mL or the chitosan nanoparticles loaded with the same amount of tea polyphenols and non-loaded chitosan nanoparticles (untreated cells were used as controls), the cells were washed with 0.1 M PBS, trypsinized, and fixed in 75% ethanol. The HepG2 cells were stained with EGFP-tagged annexin V for 20 min at room temperature and 15 μg/mL propidium iodide (PI) was also added. The HepG2 cells after treatment were detected using flow cytometry (BD Biosciences, USA) and analyzed using the CELLQuest Version 3.3 software.
2.4. Cytotoxicity assay 2.8. Analysis of cell cycle distribution The MTT [3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide blue-indicator dye]-based assay, a simple nonradioactive colorimetric assay, was used to measure cell cytotoxicity, proliferation or viability. HepG2 cells were used for the analysis of cytotoxicity in vitro. 100 μL HepG2 cells (1 × 105 cells/mL) were placed into 96-well tissueculture plates and incubated at 37 °C. After 24 h, the cells were treated with different concentrations of TP (1.0 mg/mL, 0.5 mg/mL and
Human hepatoma HepG2 cells were seeded into 24-well plate at a density of 1 × 106 cells/well. After 6 h incubation, the cells were treated with 1.0 mg/mL of TP, CNPs and TP-CNPs (which contain the same amount of TP compared with TP group), and then harvested after 15 h of exposure. Treated HepG2 cells were resuspended in 100 μL PBS, and then trypsinized, and fixed in 75% ethanol at 4 °C overnight. The
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cells were centrifuged at 2000 rpm for 3 min, and then discarded the supernatant and washed with PBS, centrifuged again, digested with RNase for 15 min, pulsed stained with propidium iodide for 10 min at room temperature, and then measured by the flow cytometry (BD Biosciences, USA). 3. Results and discussion 3.1. Characteristics of TP-CNPs The structure and size distribution of TP-CNPs and CNPs in prepared suspension are illustrated in Fig. 1. The morphology of the CNPs without TP is characterized by a spherical structure and relatively uniform surface with a uniform particle size of around 400 ± 52 nm. However, the nanoclusters that composed of TP-CNPs showed a branch shape and heterogeneous distribution. The microstructural features of TPCNPs were consistent with the description reported by Tang et al. [14]. Moreover, the mean diameter of the combined TP-CNP nanoclusters was larger than the nanoclusters of CNPs. This could be due to the ionic gelation interaction between the carboxymethyl chitosan and chitosan hydrochloride and aggregation among the CNPs after loading with TP, which resulted in the broader size distribution and heterogeneous shape of TP-CNPs. In addition, the loading performance and the control release characteristics of nanoparticles have been described in our previous report [12]. The drug content and encapsulation rate of TP-CNPs were 16% and 83% respectively. 3.2. Cytotoxicity of TP-CNPs The cytotoxicities of the TP and TP-CNPs on HepG2 cells at different intervals were evaluated through an MTT assay. Fig. 2 shows that the growth of HepG2 cells was subjected to different degrees of inhibition after 48 h treatment using TP and TP-CNP samples at different concentrations, and represented different mass concentrations of the dose–effect relationship. A comparison of the proliferation inhibition rate of TP and TP-CNPs at the same concentration can be seen from Fig. 2, the TP-CNPs suppressed at a relatively low inhibition rate compared to TP. However, with the increasing concentration of TP-CNPs,
Fig. 2. Cytotoxic effects of TP-CNPs, CNPs and TP in HepG2 cells. HepG2 cells (1 × 105 cells/mL) were cultured with different concentration of TP-CNPs, CNPs and TP for 48 h. All the data were obtained from three independent experiments (p b 0.05).
the proliferation inhibition rate was also gradually enhanced. It can therefore be concluded that the TP coated with CNPs showed a slowrelease but significant inhibitory effect against cancer cells in vitro. This phenomenon indicated that the prepared TP-CNPs can effectively inhibit the proliferation of tumor cells. Previous reports show that CNPs can enhance the intestinal absorption of the green tea catechin in mice [15,16], however, the shape and particle size of CNPs are also closely related to their delivery effect [17,18]. So there was a necessity to prepare proper CNPs for the delivery of TP. Compared to microcapsules, nanoparticles act as an effective delivery carrier owing to their smaller particle size, and suitable for the delivery of antitumor drugs. Some reports showed that the CNPs not only has the targeted delivery capability for encapsulated drugs [19], but also could enhance their bioavailability [20]. In previous reports from our laboratory, the prepared CNPs, which are fabricated using the carboxymethyl chitosan and chitosan hydrochloride biomaterials, showed good controlled release characteristics under in vitro conditions [11,12]. These results suggested that the CNPs could be used for enhancing the bioavailability of TP. 3.3. Effect of TP-CNPs on nuclei morphology The nuclei morphology changes of cancer cells treated with nanoparticles were observed using fluorescence microscopes. Fig. 3 shows
Fig. 1. (a) The TEM image and (b) Particle size distribution of TP-CNPs and CNPs in prepared suspension.
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Fig. 3. The image of HepG2 tumor cells by an inverted microscope in every group (×200) (a) Normal group, (b). TP group, (c) CNP group, and (d). TP-NP group. Scale bar: 50 μm.
Fig. 4. Ultrastructural morphology in HepG2 cells treated with TP, CNPs and TP-CNPs by TEM (magnification 10,000). (a) Normal group, (b) TP group, (c) CNP group, and (d) TP-CNP group.
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the representative microscopic images of HepG2 cells after treatment with TP-CNPs, CNPs and TP at a concentration equivalent to 1.0 mg/ mL for 48 h. The tumor cells of control group (Fig. 3a) exhibited an elongated spindle-like shape, clear boundaries between cells and vigorous growth after incubation. The cells treated with TP (Fig. 3b) showed shrinkage, round shape, and deep nuclear staining pattern. The majority of cells exhibited typical apoptotic morphology. The cells treated with CNPs exhibited shrinkage compared to control group, however, a small portion of the cells became round (Fig. 3c). In the case of tumor cells treated with TP-CNPs, the cells showed apoptotic morphology with the concentrated cytoplasm and dense chromatin in the nucleus as shown in Fig. 3d. Compared to the TP group, the cells treated with TP-CNPs showed a relatively low degree of apoptosis, which could be due to the slow release of TP in CNPs. 3.4. Effect of TP-CNPs on ultrastructure morphology Representative TEM images (Fig. 4) show the ultrastructural changes of the HepG2 cells incubated with TP, CNPs and TP-CNPs for 24 h. Fig. 3a shows the nucleus of untreated HepG2 cells with clearly defined ultrastructure, and evenly distributed chromatin. Also, the cell membrane surface possessed densely distributed microvilli. Whereas the cells treated with TP exhibited larger vacuoles, and the microvilli disappeared (Fig. 4b). The TEM images of the cells treated with CNPs also showed intact cell morphology, and no significant difference was observed when compared with the control group (Fig. 4c). However, the cancer cells treated with TP-CNPs exhibited the characteristic ultrastructural features of apoptosis, such as microvilli disappearance, margination, intracytoplasmic vacuoles, and the mitochondrial swelling with the formation of apoptotic bodies as shown in Fig. 4d. Li et al. [13] reported that paclitaxel-loaded chitosan nanoparticles can be internalized into cells and release the drug into the cytoplasm directly, thereby resulting in cancer cell apoptosis. It also can be inferred from these observations that TP-CNPs may have the potential to internalize into cells and then release of TP could induce cancer cell apoptosis.
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3.5. Cell apoptosis studies The annexin V/PI method can be used to detect the early apoptotic cells. It is ideal for the detection of cell apoptosis quantitatively with the characteristics of simplicity, sensitivity and specificity. The propidium iodide (PI) is a non-specific DNA intercalating agent, which can be used to distinguish necrotic cells from apoptotic and living cells. If the cell membrane has an injury, the DNA of the cell may emit red fluorescence when stained with PI, while the intact cell membrane does emit no red fluorescence. Therefore, the early apoptotic cells and living cells have exhibited no red fluorescence signal. The lower left quadrant represents the living cells, and the upper right quadrant shows the late apoptotic cells, while the lower right quadrant shows the early apoptotic cells in the scatterplot of bivariate flow cytometry. Fig. 5 shows the apoptosis rate of HepG2 cells treated with TP, CNPs, and TP-CNPs for 24 h. Fig. 5a represents the control group, and Fig. 5b is the group treated with TP. It was apparent from the two figures that the cell necrosis and apoptosis of TP group were significantly increased from 2.89% and 2.44% in the control group to 64.29% and 31.68%, respectively. While the number of living cells decreased significantly, almost no living cell could be found. The results showed that TP has a strong antitumor activity and could induce the necrosis and apoptosis of tumor cells. Fig. 5c shows that the rates of cell necrosis and cell apoptosis were increased compared to the control group, but there was no significant difference. It indicated that the rates of necrosis and apoptosis of tumor cells induced by CNPs were relatively weak. Fig. 5d shows the rates of necrosis and apoptosis of HepG2 cells treated with TP-CNPs after 24 h. The figure represents a little change in cell necrosis rate when compared to the control group, while the apoptosis rate was increased to 23.83%. It was indicated that the antitumor activity of TP-CNPs was mainly induced through cell apoptosis. Comparing the treatment results of TP with TP-CNPs, TP can induce tumor cell apoptosis, and also could promote cell necrosis. The necrotic effect of CNPs was much higher than that of TP-CNPs as shown in Fig. 5. The TP-CNPs induced cancer cell apoptosis mainly through constant release of TP
Fig. 5. TP, CNPs and TP-CNPs induced apoptosis. (a) Pictures were representative results from flow cytometry of propidiumiodine (PI)-stained HepG2 cells treated with 1.0 mg/mL of tea polyphenols, plain CNPs or TP-CNPs (which contain tea polyphenols at the same concentration with tea polyphenol group) for 24 h.
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from CNPs, and the cancer cell membrane showed integrity. While the cell necrosis caused by CNPs mainly manifested in the cell membrane damage and resultant enzyme leakage [21]. In addition, the CNPs themselves in TP-CNPs have showed a limited anticancer activity than TP. Therefore, TP-CNPs could exhibit relatively stronger apoptosis and weaker necrosis effects on tumor cells than CNPs. 3.6. Cell cycle analysis Flow cytometry can detect the percentage of different cells M1, M2, and M3, corresponding to the cells sub-G1, G0/G1 phase and S phase. In this study, the impact of TP-CNPs on HepG2 tumor cell cycle progression and distribution was investigated by flow cytometry. Fig. 6 shows the cell cycle changes of HepG2 cells treated with TP, CNPs and TP-CNPs after 24 h. Compared to the control group, the cells treated with TP have exhibited increased in G0/G1 phase, and the cell cycle progression was increased to 23.75%, while the S phase was reduced to 17.99% (Fig 6b). The group treated with CNPs has shown a mild change in the cell cycle progression (Fig 6c), and a slight increase could be seen only in G0/G1 phase and S phase cells. Comparing the group of TP-CNPs (Fig 6d) with the control group, the number of cells in G0/G1 phase was increased to 5.54%, while the total cells in G1 and S phase were decreased to some extent. Some reports also showed that TP can induce cell cycle arrest in G0/G1 phase and trigger cell death by apoptotic mechanism [22–25]. In addition, we previously reported that the TP from TP-CNPs can release in a continuous manner for up to 48 h [12]. Therefore, these results indicated that CNPs have the potential to use as the anticancer drug carriers. 4. Conclusion CNPs have become attractive for their promising properties in the aspect of drug delivery systems during recent years. In this study, the CNPs prepared by using two different chitosan biomaterials could be considered as an effective carrier for the delivery of TP. The antitumor effects of TP loaded CNPs on HepG2 cells have been manifested by
cytotoxicity, cell morphology analysis, apoptosis detection and cell cycle measurement. The results indicated that TP-CNPs can inhibit the proliferation of HepG2, and their antitumor effect was mainly achieved through necrosis and apoptosis induction in cancer cells. Compared to TP, the CNPs themselves showed limited antitumor activities and their inhibition mechanism was mainly through inducing tumor cell necrosis. Further research will focus on the comparison of the antitumor effects between TP-CNPs and TP using animal model. Acknowledgments This work was supported by the National Natural Science Foundation of China (30871743), the 111 Project of Education Ministry of China (B07030), and the National High Technology Research and Development Program of China (2007AA100403). The authors would like to thank Dr. Pradeep Puligundla for refining the language. References [1] N. Hail Jr., M. Cortes, E.N. Drake, J.E. Spallholz, Free Radic. Biol. Med. 45 (2008) 97–110. [2] C.S. Yang, J.D. Lambert, S. Sang, Arch. Toxicol. 83 (2009) 11–21. [3] D. Chen, Q.P. Dou, Int. J. Mol. Sci. 9 (2008) 1196–1206. [4] S.M. Henning, Y. Niu, Y. Liu, N.H. Lee, Y. Hara, G.D. Thames, R.R. Minutti, C.L. Carpenter, H. Wang, D. Heber, J. Nutr. Biochem. 16 (2005) 610–616. [5] Y. Yuan, C. Liu, J. Qian, J. Wang, Y. Zhang, Biomaterials 31 (2010) 730–740. [6] H.Y. Hwang, I.S. Kim, I.C. Kwon, Y.H. Kim, J. Control. Release 128 (2008) 23–31. [7] H. Luo, J. Li, X. Chen, Biomed. Pharmacother. 64 (2010) 521–526. [8] W. Ajun, S. Yan, G. Li, L. Huili, Carbohydr. Polym. 75 (2009) 566–574. [9] A.W. Pan, B.B. Wu, J.M. Wu, Chin. Chem. Lett. 20 (2009) 79–83. [10] L. Qi, Z. Xu, X. Jiang, Y. Li, M. Wang, Bioorg. Med. Chem. Lett. 15 (2005) 1397–1399. [11] J. Liang, F. Li, Y. Fang, W. Yang, X. An, L. Zhao, Z. Xin, Q. Hu, Eur. Food Res. Technol. 231 (2010) 917–924. [12] J. Liang, F. Li, Y. Fang, W. Yang, X. An, L. Zhao, Z. Xin, L. Cao, Q. Hu, Colloids Surf. B: Biointerfaces 82 (2011) 297–301. [13] F. Li, J. Li, X. Wen, S. Zhou, X. Tong, P. Su, H. Li, D. Shi, Mater. Sci. Eng. 29 (2009) 2392–2397. [14] D.W. Tang, S.H. Yu, Y.C. Ho, B.Q. Huang, G.J. Tsai, H.-Y. Hsieh, H.W. Sung, F.L. Mi, Food Hydrocoll. 30 (2013) 33–41. [15] A. Dube, J.A. Nicolazzo, I. Larson, Eur. J. Pharm. Sci. 41 (2010) 219–225. [16] A. Dube, J.A. Nicolazzo, I. Larson, Eur. J. Pharm. Sci. 44 (2011) 422–426.
Fig. 6. TP, CNPs and TP-CNPs induced cell apoptosis. The picture (a) was normal HepG2 cell, and the pictures (b), (c), and (d) were representative results from flow cytometry of propidiumiodine (PI)-stained HepG2 cells treated with 1.0 mg/mL of TP, CNPs or TP-CNPs for 24 h.
J. Liang et al. / Materials Science and Engineering C 36 (2014) 7–13 [17] S.A. Agnihotri, N.N. Mallikarjuna, T.M. Aminabhavi, J. Control. Release 100 (2004) 5–28. [18] C. He, L. Yin, C. Tang, C. Yin, Biomaterials 33 (2012) 8569–8578. [19] O.C. Farokhzad, R. Langer, ACS Nano 3 (2009) 16–20. [20] B. Hu, Y. Ting, X. Yang, W. Tang, X. Zeng, Q. Huang, Chem. Commun. 48 (2012) 2421–2423. [21] J.W. Loh, G. Yeoh, M. Saunders, L.Y. Lim, Toxicol. Appl. Pharm. 249 (2010) 148–157.
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[22] N. Ahmad, D.K. Feyes, A.L. Nieminen, R. Agarwal, H. Mukhtar, J. Natl Cancer Inst. 89 (1997) 1881–1886. [23] S. Gupta, T. Hussain, H. Mukhtar, Arch. Biochem. Biophys. 410 (2003) 177–185. [24] N. Ahmad, P.Y. Cheng, H. Mukhtar, Biochem. Biophys. Res. Commun. 275 (2000) 328–334. [25] J.D. Lambert, C.S. Yang, Mutat. Res. Fundam. Mol. Mech. Mutagen. 523 (2003) 201–208.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells Jin Liang a,b, Feng Li b, Yong Fang c, Wenjian Yang c, Xinxin An b, Liyan Zhao b, Zhihong Xin b, Lin Cao b, Qiuhui Hu b,c,⁎ a b c
Key Laboratory of Tea Biochemistry and Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, People's Republic of China College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People's Republic of China College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023, People's Republic of China
a r t i c l e
i n f o
Article history: Received 26 November 2012 Received in revised form 24 October 2013 Accepted 26 November 2013 Available online 5 December 2013 Keywords: Tea polyphenols Chitosan Nanoparticles Hepatoma Apoptosis
a b s t r a c t Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenolloaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs. © 2013 Published by Elsevier B.V.
1. Introduction Nowadays, cancer is one of the major dread diseases and chemoprevention is considered a valid approach to reduce the incidence of cancer [1]. Tea polyphenols (TP) from the dried leaves of the plant Camellia sinensis are known to possess strong antioxidant and anti-carcinogenic activities [2]. Although TP are widely used for the prevention and treatment of cancer, the therapeutic effects are limited due to their certain drawbacks, such as poor stability in gastrointestinal tract and limited bioavailability in vivo [3]. It has been reported that the biological activity of TP might depend on the form of their administration [4]. Therefore, a lot of research has been directed towards the development of effective methods to overcome the bottlenecks. Nanoparticles, especially those with highly controlled shapes, sizes and some unique physical and ⁎ Corresponding author at: College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People's Republic of China. Tel./fax: + 86 25 84399086. E-mail address: [email protected] (Q. Hu). 0928-4931/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.msec.2013.11.039
chemical properties including the antitumor activities [5], have been studied extensively as drug carriers for the improvement of the bioavailability of drug with poor absorption characteristics. Owing to their small size, prolonged circulation time, and sustained drug release profile, nano-sized polymeric nanoparticles bearing anticancer drugs have received increased attention for their ability to improve the efficacy of anticancer drugs [6]. Nanoparticles can facilitate controlled and targeted drug delivery of the encapsulated anticancer drugs with high efficacy and low side effects [7]. They not only have the ability to overcome biological barriers and accumulate preferentially in tumors, but also can recognize single cancer cells, which are useful to the detection and treatment of cancer. Chitosan, a deacetylated derivative of chitin, has been applied extensively as a functional biopolymer in food and pharmaceutical industry, and is well known for its abundant, renewable, non-toxic and biodegradable nature. Because of its unique chemical structure, chitosan has been investigated extensively in the development of controlled release drug delivery systems. Especially, the mucoadhesive property of chitosan can enhance drug transmucosal absorption and promote its
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J. Liang et al. / Materials Science and Engineering C 36 (2014) 7–13
sustained release [8,9]. It has been reported that chitosan nanoparticles as drug carriers with small particle size and positive surface charge can exhibit a higher antitumor activity [10]. Our previous studies showed that the tea polyphenol-loaded chitosan nanoparticles (TP-CNPs) could be obtained by ionic gelation using two biocompatible materials, namely N-carboxymethyl chitosan and chitosan hydrochloride, and the optimization of particle size and entrapment efficiency have been discussed [11]. However, the antitumor biological activity of TP-CNPs is still unknown. Therefore, in this study, the antitumor effects of the TP-CNP, TP and chitosan nanoparticles (CNPs) have been evaluated using HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, apoptosis detection, and cell cycle measurement, and the antitumor mechanism was also hypothesized. 2. Materials and methods
0.25 mg/mL) and TP-CNPs loaded with the same amount of TP. The untreated cells were used as the control. The plates were incubated in a humidified 5% CO2 balanced-air incubator at 37 °C for 48 h. Then 10 μL of 5 mg/mL MTT solution was added into each well and the plates were incubated for another 4 h, and then the medium was discarded. Dimethyl sulfoxide (100 μL) was added into each well of the 96-well plate, and the solution was vigorously mixed to dissolve tetrazolium dye. The absorbance of each well was measured by an enzyme-linked immunosorbent assay reader (BioTek; Austria) at a test wavelength of 570 nm and the cell cytotoxicity rate was calculated by the following equation: Cell cytotoxicity ¼ ½1–T=C 100% where C is the number of viable cells after 48 h of incubation without nanoparticles and T is the number of viable cells after 48 h of incubation with nanoparticles.
2.1. Materials 2.5. Morphological examination using microscope TP extract with a purity of 93% was obtained from dried green tea leaves by the solvent extraction method using 50% ethanol and then purified with H1020 resins (Nankai University Chemical Plant, Tianjin, China). HepG2 cells were obtained from Nanjing University (Nanjing, Jiangsu, China). All the other reagents used were of analytical grade.
100 μL HepG2 cells (1 × 105 cells/mL) were seeded on 96-well culture plates and TP-CNPs, CNPs and TP (1.0 mg/mL) were added into the cells, and after 48 h incubation, morphological changes of cells were examined under a fluorescent microscope (CKX41, Olympus Corporation, Tokyo, Japan).
2.2. The preparation of TP-CNPs 2.6. Transmission electron microscopy assay The TP-CNPs were prepared according to our previously reported optimized conditions [11,12]. Briefly, N-carboxymethyl chitosan (Mv = 61 kDa, Degree of deacetylation 83%) and chitosan hydrochloride (Mv = 90 kDa, Degree of deacetylation 85%) were dissolved in distilled water and the solutions of different concentrations were prepared by adding the extracts of TP into carboxymethyl chitosan solution. The optimal levels of carboxymethyl chitosan concentration, chitosan hydrochloride concentration and the amount of TP were 3.63 mg/mL, 1.19 mg/mL, and 10.94 mg, respectively. The main preparation process was the following steps. First, TP was added into the chitosan hydrochloride solution (30 mL), and then the carboxymethyl chitosan solution (12 mL) was added dropwise into the chitosan hydrochloride and TP mixed solution under stirring at room temperature, and continuously stirred for 30 min. The formation of nanoclusters was based on an ionic gelation interaction between positively-charged amine groups of chitosan hydrochloride chitosan and negatively-charged carboxyl groups of carboxymethyl chitosan. The nanocluster suspensions were immediately subjected to further analysis for their structure and size distribution characterization. The nanocluster powders as experimental samples were obtained by high speed centrifugation (12,000 rpm, 30 min) followed by freeze-drying process. Meanwhile, the CNPs without TP were prepared to use as the control group. 2.3. Cell culture Human hepatoma HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) equilibrated with 5% CO2 and 95% air at 37 °C. The medium was supplemented with 10% fetal calf serum (FCS), 50 mg/L streptomycin and 75 mg/L penicillin sulfate.
Changes in the ultrastructure of HepG2 cells after treatment with TPCNPs were observed using the transmission electron microscope (TEM) according to the previously published protocol [7]. Briefly, after incubation with TP-CNPs (1 mg/mL) at 37 °C for 24 h, HepG2 cells were collected by centrifugation and washed twice with PBS, and then fixated immediately in Karnovsky solution (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na cacodylate/HCl, pH 7.4). The postfixation was performed in 1% OsO4 with 1.5% potassium ferrocyanide, followed by dehydration and drying. After being trimmed, mounted and coated, the ultrathin sections were observed and photographed by using the TEM (JEOL H-7650, Hitachi High-Technologies Corporation, Tokyo, Japan). At the same time, the TEM images of TP and CNPs were also captured using the same method. 2.7. Determination of cell apoptosis The cell apoptosis was detected by annexin V/PI double stain assay. The annexin V/PI staining was performed as described previously [13]. Briefly, HepG2 cells (3 × 105 cells/well) were seeded in 24-well plates and incubated at 37 °C in a 5% CO2 incubator overnight. After treatment with tea polyphenols at 1.0 mg/mL or the chitosan nanoparticles loaded with the same amount of tea polyphenols and non-loaded chitosan nanoparticles (untreated cells were used as controls), the cells were washed with 0.1 M PBS, trypsinized, and fixed in 75% ethanol. The HepG2 cells were stained with EGFP-tagged annexin V for 20 min at room temperature and 15 μg/mL propidium iodide (PI) was also added. The HepG2 cells after treatment were detected using flow cytometry (BD Biosciences, USA) and analyzed using the CELLQuest Version 3.3 software.
2.4. Cytotoxicity assay 2.8. Analysis of cell cycle distribution The MTT [3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide blue-indicator dye]-based assay, a simple nonradioactive colorimetric assay, was used to measure cell cytotoxicity, proliferation or viability. HepG2 cells were used for the analysis of cytotoxicity in vitro. 100 μL HepG2 cells (1 × 105 cells/mL) were placed into 96-well tissueculture plates and incubated at 37 °C. After 24 h, the cells were treated with different concentrations of TP (1.0 mg/mL, 0.5 mg/mL and
Human hepatoma HepG2 cells were seeded into 24-well plate at a density of 1 × 106 cells/well. After 6 h incubation, the cells were treated with 1.0 mg/mL of TP, CNPs and TP-CNPs (which contain the same amount of TP compared with TP group), and then harvested after 15 h of exposure. Treated HepG2 cells were resuspended in 100 μL PBS, and then trypsinized, and fixed in 75% ethanol at 4 °C overnight. The
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cells were centrifuged at 2000 rpm for 3 min, and then discarded the supernatant and washed with PBS, centrifuged again, digested with RNase for 15 min, pulsed stained with propidium iodide for 10 min at room temperature, and then measured by the flow cytometry (BD Biosciences, USA). 3. Results and discussion 3.1. Characteristics of TP-CNPs The structure and size distribution of TP-CNPs and CNPs in prepared suspension are illustrated in Fig. 1. The morphology of the CNPs without TP is characterized by a spherical structure and relatively uniform surface with a uniform particle size of around 400 ± 52 nm. However, the nanoclusters that composed of TP-CNPs showed a branch shape and heterogeneous distribution. The microstructural features of TPCNPs were consistent with the description reported by Tang et al. [14]. Moreover, the mean diameter of the combined TP-CNP nanoclusters was larger than the nanoclusters of CNPs. This could be due to the ionic gelation interaction between the carboxymethyl chitosan and chitosan hydrochloride and aggregation among the CNPs after loading with TP, which resulted in the broader size distribution and heterogeneous shape of TP-CNPs. In addition, the loading performance and the control release characteristics of nanoparticles have been described in our previous report [12]. The drug content and encapsulation rate of TP-CNPs were 16% and 83% respectively. 3.2. Cytotoxicity of TP-CNPs The cytotoxicities of the TP and TP-CNPs on HepG2 cells at different intervals were evaluated through an MTT assay. Fig. 2 shows that the growth of HepG2 cells was subjected to different degrees of inhibition after 48 h treatment using TP and TP-CNP samples at different concentrations, and represented different mass concentrations of the dose–effect relationship. A comparison of the proliferation inhibition rate of TP and TP-CNPs at the same concentration can be seen from Fig. 2, the TP-CNPs suppressed at a relatively low inhibition rate compared to TP. However, with the increasing concentration of TP-CNPs,
Fig. 2. Cytotoxic effects of TP-CNPs, CNPs and TP in HepG2 cells. HepG2 cells (1 × 105 cells/mL) were cultured with different concentration of TP-CNPs, CNPs and TP for 48 h. All the data were obtained from three independent experiments (p b 0.05).
the proliferation inhibition rate was also gradually enhanced. It can therefore be concluded that the TP coated with CNPs showed a slowrelease but significant inhibitory effect against cancer cells in vitro. This phenomenon indicated that the prepared TP-CNPs can effectively inhibit the proliferation of tumor cells. Previous reports show that CNPs can enhance the intestinal absorption of the green tea catechin in mice [15,16], however, the shape and particle size of CNPs are also closely related to their delivery effect [17,18]. So there was a necessity to prepare proper CNPs for the delivery of TP. Compared to microcapsules, nanoparticles act as an effective delivery carrier owing to their smaller particle size, and suitable for the delivery of antitumor drugs. Some reports showed that the CNPs not only has the targeted delivery capability for encapsulated drugs [19], but also could enhance their bioavailability [20]. In previous reports from our laboratory, the prepared CNPs, which are fabricated using the carboxymethyl chitosan and chitosan hydrochloride biomaterials, showed good controlled release characteristics under in vitro conditions [11,12]. These results suggested that the CNPs could be used for enhancing the bioavailability of TP. 3.3. Effect of TP-CNPs on nuclei morphology The nuclei morphology changes of cancer cells treated with nanoparticles were observed using fluorescence microscopes. Fig. 3 shows
Fig. 1. (a) The TEM image and (b) Particle size distribution of TP-CNPs and CNPs in prepared suspension.
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Fig. 3. The image of HepG2 tumor cells by an inverted microscope in every group (×200) (a) Normal group, (b). TP group, (c) CNP group, and (d). TP-NP group. Scale bar: 50 μm.
Fig. 4. Ultrastructural morphology in HepG2 cells treated with TP, CNPs and TP-CNPs by TEM (magnification 10,000). (a) Normal group, (b) TP group, (c) CNP group, and (d) TP-CNP group.
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the representative microscopic images of HepG2 cells after treatment with TP-CNPs, CNPs and TP at a concentration equivalent to 1.0 mg/ mL for 48 h. The tumor cells of control group (Fig. 3a) exhibited an elongated spindle-like shape, clear boundaries between cells and vigorous growth after incubation. The cells treated with TP (Fig. 3b) showed shrinkage, round shape, and deep nuclear staining pattern. The majority of cells exhibited typical apoptotic morphology. The cells treated with CNPs exhibited shrinkage compared to control group, however, a small portion of the cells became round (Fig. 3c). In the case of tumor cells treated with TP-CNPs, the cells showed apoptotic morphology with the concentrated cytoplasm and dense chromatin in the nucleus as shown in Fig. 3d. Compared to the TP group, the cells treated with TP-CNPs showed a relatively low degree of apoptosis, which could be due to the slow release of TP in CNPs. 3.4. Effect of TP-CNPs on ultrastructure morphology Representative TEM images (Fig. 4) show the ultrastructural changes of the HepG2 cells incubated with TP, CNPs and TP-CNPs for 24 h. Fig. 3a shows the nucleus of untreated HepG2 cells with clearly defined ultrastructure, and evenly distributed chromatin. Also, the cell membrane surface possessed densely distributed microvilli. Whereas the cells treated with TP exhibited larger vacuoles, and the microvilli disappeared (Fig. 4b). The TEM images of the cells treated with CNPs also showed intact cell morphology, and no significant difference was observed when compared with the control group (Fig. 4c). However, the cancer cells treated with TP-CNPs exhibited the characteristic ultrastructural features of apoptosis, such as microvilli disappearance, margination, intracytoplasmic vacuoles, and the mitochondrial swelling with the formation of apoptotic bodies as shown in Fig. 4d. Li et al. [13] reported that paclitaxel-loaded chitosan nanoparticles can be internalized into cells and release the drug into the cytoplasm directly, thereby resulting in cancer cell apoptosis. It also can be inferred from these observations that TP-CNPs may have the potential to internalize into cells and then release of TP could induce cancer cell apoptosis.
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3.5. Cell apoptosis studies The annexin V/PI method can be used to detect the early apoptotic cells. It is ideal for the detection of cell apoptosis quantitatively with the characteristics of simplicity, sensitivity and specificity. The propidium iodide (PI) is a non-specific DNA intercalating agent, which can be used to distinguish necrotic cells from apoptotic and living cells. If the cell membrane has an injury, the DNA of the cell may emit red fluorescence when stained with PI, while the intact cell membrane does emit no red fluorescence. Therefore, the early apoptotic cells and living cells have exhibited no red fluorescence signal. The lower left quadrant represents the living cells, and the upper right quadrant shows the late apoptotic cells, while the lower right quadrant shows the early apoptotic cells in the scatterplot of bivariate flow cytometry. Fig. 5 shows the apoptosis rate of HepG2 cells treated with TP, CNPs, and TP-CNPs for 24 h. Fig. 5a represents the control group, and Fig. 5b is the group treated with TP. It was apparent from the two figures that the cell necrosis and apoptosis of TP group were significantly increased from 2.89% and 2.44% in the control group to 64.29% and 31.68%, respectively. While the number of living cells decreased significantly, almost no living cell could be found. The results showed that TP has a strong antitumor activity and could induce the necrosis and apoptosis of tumor cells. Fig. 5c shows that the rates of cell necrosis and cell apoptosis were increased compared to the control group, but there was no significant difference. It indicated that the rates of necrosis and apoptosis of tumor cells induced by CNPs were relatively weak. Fig. 5d shows the rates of necrosis and apoptosis of HepG2 cells treated with TP-CNPs after 24 h. The figure represents a little change in cell necrosis rate when compared to the control group, while the apoptosis rate was increased to 23.83%. It was indicated that the antitumor activity of TP-CNPs was mainly induced through cell apoptosis. Comparing the treatment results of TP with TP-CNPs, TP can induce tumor cell apoptosis, and also could promote cell necrosis. The necrotic effect of CNPs was much higher than that of TP-CNPs as shown in Fig. 5. The TP-CNPs induced cancer cell apoptosis mainly through constant release of TP
Fig. 5. TP, CNPs and TP-CNPs induced apoptosis. (a) Pictures were representative results from flow cytometry of propidiumiodine (PI)-stained HepG2 cells treated with 1.0 mg/mL of tea polyphenols, plain CNPs or TP-CNPs (which contain tea polyphenols at the same concentration with tea polyphenol group) for 24 h.
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from CNPs, and the cancer cell membrane showed integrity. While the cell necrosis caused by CNPs mainly manifested in the cell membrane damage and resultant enzyme leakage [21]. In addition, the CNPs themselves in TP-CNPs have showed a limited anticancer activity than TP. Therefore, TP-CNPs could exhibit relatively stronger apoptosis and weaker necrosis effects on tumor cells than CNPs. 3.6. Cell cycle analysis Flow cytometry can detect the percentage of different cells M1, M2, and M3, corresponding to the cells sub-G1, G0/G1 phase and S phase. In this study, the impact of TP-CNPs on HepG2 tumor cell cycle progression and distribution was investigated by flow cytometry. Fig. 6 shows the cell cycle changes of HepG2 cells treated with TP, CNPs and TP-CNPs after 24 h. Compared to the control group, the cells treated with TP have exhibited increased in G0/G1 phase, and the cell cycle progression was increased to 23.75%, while the S phase was reduced to 17.99% (Fig 6b). The group treated with CNPs has shown a mild change in the cell cycle progression (Fig 6c), and a slight increase could be seen only in G0/G1 phase and S phase cells. Comparing the group of TP-CNPs (Fig 6d) with the control group, the number of cells in G0/G1 phase was increased to 5.54%, while the total cells in G1 and S phase were decreased to some extent. Some reports also showed that TP can induce cell cycle arrest in G0/G1 phase and trigger cell death by apoptotic mechanism [22–25]. In addition, we previously reported that the TP from TP-CNPs can release in a continuous manner for up to 48 h [12]. Therefore, these results indicated that CNPs have the potential to use as the anticancer drug carriers. 4. Conclusion CNPs have become attractive for their promising properties in the aspect of drug delivery systems during recent years. In this study, the CNPs prepared by using two different chitosan biomaterials could be considered as an effective carrier for the delivery of TP. The antitumor effects of TP loaded CNPs on HepG2 cells have been manifested by
cytotoxicity, cell morphology analysis, apoptosis detection and cell cycle measurement. The results indicated that TP-CNPs can inhibit the proliferation of HepG2, and their antitumor effect was mainly achieved through necrosis and apoptosis induction in cancer cells. Compared to TP, the CNPs themselves showed limited antitumor activities and their inhibition mechanism was mainly through inducing tumor cell necrosis. Further research will focus on the comparison of the antitumor effects between TP-CNPs and TP using animal model. Acknowledgments This work was supported by the National Natural Science Foundation of China (30871743), the 111 Project of Education Ministry of China (B07030), and the National High Technology Research and Development Program of China (2007AA100403). The authors would like to thank Dr. Pradeep Puligundla for refining the language. References [1] N. Hail Jr., M. Cortes, E.N. Drake, J.E. Spallholz, Free Radic. Biol. Med. 45 (2008) 97–110. [2] C.S. Yang, J.D. Lambert, S. Sang, Arch. Toxicol. 83 (2009) 11–21. [3] D. Chen, Q.P. Dou, Int. J. Mol. Sci. 9 (2008) 1196–1206. [4] S.M. Henning, Y. Niu, Y. Liu, N.H. Lee, Y. Hara, G.D. Thames, R.R. Minutti, C.L. Carpenter, H. Wang, D. Heber, J. Nutr. Biochem. 16 (2005) 610–616. [5] Y. Yuan, C. Liu, J. Qian, J. Wang, Y. Zhang, Biomaterials 31 (2010) 730–740. [6] H.Y. Hwang, I.S. Kim, I.C. Kwon, Y.H. Kim, J. Control. Release 128 (2008) 23–31. [7] H. Luo, J. Li, X. Chen, Biomed. Pharmacother. 64 (2010) 521–526. [8] W. Ajun, S. Yan, G. Li, L. Huili, Carbohydr. Polym. 75 (2009) 566–574. [9] A.W. Pan, B.B. Wu, J.M. Wu, Chin. Chem. Lett. 20 (2009) 79–83. [10] L. Qi, Z. Xu, X. Jiang, Y. Li, M. Wang, Bioorg. Med. Chem. Lett. 15 (2005) 1397–1399. [11] J. Liang, F. Li, Y. Fang, W. Yang, X. An, L. Zhao, Z. Xin, Q. Hu, Eur. Food Res. Technol. 231 (2010) 917–924. [12] J. Liang, F. Li, Y. Fang, W. Yang, X. An, L. Zhao, Z. Xin, L. Cao, Q. Hu, Colloids Surf. B: Biointerfaces 82 (2011) 297–301. [13] F. Li, J. Li, X. Wen, S. Zhou, X. Tong, P. Su, H. Li, D. Shi, Mater. Sci. Eng. 29 (2009) 2392–2397. [14] D.W. Tang, S.H. Yu, Y.C. Ho, B.Q. Huang, G.J. Tsai, H.-Y. Hsieh, H.W. Sung, F.L. Mi, Food Hydrocoll. 30 (2013) 33–41. [15] A. Dube, J.A. Nicolazzo, I. Larson, Eur. J. Pharm. Sci. 41 (2010) 219–225. [16] A. Dube, J.A. Nicolazzo, I. Larson, Eur. J. Pharm. Sci. 44 (2011) 422–426.
Fig. 6. TP, CNPs and TP-CNPs induced cell apoptosis. The picture (a) was normal HepG2 cell, and the pictures (b), (c), and (d) were representative results from flow cytometry of propidiumiodine (PI)-stained HepG2 cells treated with 1.0 mg/mL of TP, CNPs or TP-CNPs for 24 h.
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