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Apoptosis of Hepatocellular Carcinoma Cells Induced by Nanoencapsulated Polysaccharides Extracted from Antrodia Camphorata

Antrodia camphorata is a well-known medicinal mushroom in Taiwan and has been studied for decades, especially with focus on anti-cancer activity. Polysaccharides are the major bioactive compounds reported with anti-cancer activity, but the debates on how they target cells still remain. Research addressing the encapsulation of polysaccharides from A. camphorata extract (ACE) to enhance anti-cancer activity is rare. In this study, ACE polysaccharides were nano-encapsulated in chitosan-silica and silica (expressed as ACE/CS and ACE/S, respectively) to evaluate the apoptosis effect on a hepatoma cell line (Hep G2). The results showed that ACE polysaccharides, ACE/CS and ACE/S all could damage the Hep G2 cell membrane and cause cell death, especially in the ACE/CS group. In apoptosis assays, DNA fragmentation and sub-G1 phase populations were increased, and the mitochondrial membrane potential decreased significantly after treatments. ACE/CS and ACE/S could also increase reactive oxygen species (ROS) generation, induce Fas/APO-1 (apoptosis antigen 1) expression and elevate the proteolytic activities of caspase-3, caspase-8 and caspase-9 in Hep G2 cells. Unsurprisingly, ACE/CS induced a similar apoptosis mechanism at a lower dosage (ACE polysaccharides = 13.2 μg/mL) than those of ACE/S (ACE polysaccharides = 21.2 μg/mL) and ACE polysaccharides (25 μg/mL). Therefore, the encapsulation of ACE polysaccharides by chitosan-silica nanoparticles may provide a viable approach for enhancing anti-tumor efficacy in liver cancer cells.


Introduction
Medicinal mushrooms have become increasingly popular in recent years for their potential in disease prevention, especially in tumor inhibition [1,2]. Antrodia camphorata is a well-known mushroom that has been used as an herbal medicine for centuries in Taiwan. This mushroom has been used in the treatment of various diseases; e.g., diarrhea, abdominal pain and hypertension [3,4]. Moreover, the pharmacological properties of A. camphorata, such as hepatoprotective properties [5,6] and anti-tumor activities [7][8][9], have been stated and summarized. Generally, polysaccharides and triterpenoids are the major bioactive components in medicinal mushrooms, and the polysaccharides extracted from A. camphorata were found to have anti-hepatitis B surface antigen [10] and anti-tumor effects [3,11].
Rapid development in nanotechnology engenders a variety of nanoparticles with novel bioactive ingredients delivered into cancer cells [12]. Natural and synthetic polymers as well as inorganic materials could be used for constructing functional nanoparticles. Among these materials, silica and chitosan have received more attention because of their abundance in nature. Chitosan is a cationic polysaccharide with attractive chemical and biological characteristics for pharmaceutical purposes [13] and has been incorporated into various carrier systems for drug delivery to improve their absorption and targeting characteristics [14][15][16][17][18][19][20].
Silica nanostructures allow one to encapsulate biomolecules and therefore deliver multiple clinical functions. Most researchers synthesize silica with various sizes, morphology and surface functional properties of silicon alkoxide by the sol-gel process [21,22]. In the past decade, a variety of silicate-based biohybrid materials containing silica and biopolymers have been produced and are widely used in bio-encapsulation applications due to the low environmental impact, low toxicity and good biocompatibility [23]. We recently demonstrated that chitosan was capable of forming composite nanoparticles with silica [24]. Moreover, the presence of chitosan in the composite, i.e., chitosan-silica nanoparticles, significantly reduced the cytotoxicity of silica nanoparticles [25]. Nevertheless, little information is currently available on changes in the efficacy of bioactive ingredients encapsulated by biopolymer-silica hybrid nanoparticles.
The aim of this study was to evaluate the effects of A. camphorata extract (ACE) polysaccharides, ACE polysaccharides encapsulated in chitosan-silica nanoparticles (ACE/CS) and encapsulated in silica nanoparticles (ACE/S) on apoptosis of cells in a human liver cancer cell line (Hep G2). We investigated the cell cycle, mitochondrial membrane potential, Fas/APO-1, caspase-8, caspase-9 and caspase-3 signaling molecules, which are strongly associated with the apoptosis signal transduction pathway and are related to the responses of tumor cells treated with anti-cancer compounds.

Preparations of ACE polysaccharides, ACE/CS and ACE/S
Powdered fruiting bodies of cultivated A. camphorata (approximately 70 g) were soaked in 500 mL ethanol (70%) for 12 h. The suspension was centrifuged at 5,520× g for 30 min to remove the insoluble matter, and the supernatant was concentrated with a vacuum evaporator until the volume reached 100 mL. The above solution was dialyzed (10 KDa MWCO) overnight, filtered with a filter paper (pore size 0.45 μm) and freeze-dried to powder form for further analysis. For nanoparticle preparation, the qualified chitosan samples purchased from a commercial supplier were analyzed for the degree of deacetylation (DD) up to 81% and molecular weight (Mw), which was approximately 200 kDA. ACE/CS was prepared as previously described [24]. Briefly, the sodium silicate was dissolved in 30 mL buffer (0.05 M sodium acetate solution) to prepare a 0.55% (w/w) solution (pH = 6.0). After 10 min agitation in a magnetic stirrer, 6 mL ACE polysaccharides solution (0.1% w/w) and 3 mL chitosan solution (0.55% w/w) were added. The resulting solution was mixed completely and set aside for particle synthesis (4 h) without disturbance. ACE/CS was collected by centrifugation at 5,520× g for 30 min. For ACE/ S preparation, silica solution at the same concentration described above without chitosan solution was used and ACE/S was collected after 12 h of the particle synthesis process.

Cell viability analysis
Hep G2 cells were seeded in 96-well plates at a density of 2.0 × 10 5 cells/well. After incubation for 8 h, the medium was removed and replaced by 20 μL of different concentrations of ACE polysaccharides, ACE/ES, ACE/S and nanoparticles without loading, which were suspended in deionized water and ultrasonicated for 1 h to prevent agglomeration. Controls were cells treated with an equivalent volume of serum-free medium instead of suspensions. Cells were further incubated for 24 h or 48 h and viability was assessed by using an MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium bromide) test [26]. In brief, fresh media with 100 μL of 5 mg/mL MTT stain (Sigma-Aldrich in vitro toxicology assay kit) was added into each well and incubated for 4 h. The media/stain was drawn out and purple color crystals were dissolved using acidic isopropyl alcohol. The absorbance in each well was measured at 570 nm using a 4294B Microplate Reader (Tecan Sunrise, American Instrument Exchange Inc. USA). All experiments were repeated 3 times independently to ensure reproducibility and data were acquired in triplicate (n = 3). Cell viability was calculated with the following formula: Where control stands for culture without any treatment.

Lactate dehydrogenase release assay
Cells at a density of 2.0 × 10 5 cells/well were treated in the same protocol described above except at the indicated concentrations of suspensions. After 48 h incubation, the cultured cells were centrifuged at 430× g for 5 min and the cell medium was transferred to a new 96-well plate (50 μL/well). Upon the addition of the lactate dehydrogenase (LDH) reaction mixture (Promega, CytoTox 96R non-radioactive assay kit) to the wells (50 μL/well), they were kept in the dark for 30 min and then 1 N HCl (25 μL/well) was added to each sample to terminate the reaction. The resulting absorbance was measured at 490 nm. All experiments were repeated 3 times independently to ensure reproducibility and data were acquired in triplicate (n = 3). Control and positive control experiments were performed with medium only and with 0.1% (w/v) Triton X-100, with cytotoxicity identified at 100%, respectively. LDH release (%) was calculated using following equation: Gel electrophoresis examination for DNA fragments Hep G2 cells were seeded at the density of 2.0 × 10 5 cells/mL in 10-cm dishes containing medium supplemented with 2% FBS and incubated for 4 h. Tumor necrosis factor-related apoptosisinducing ligand (Trail), for positive control, ACE polysaccharides, ACE/CS and ACE/S suspensions were added to the dishes, protected from light and incubated for 48 h. Cells were harvested by centrifugation at 250× g after incubation, lysed in buffer solution and centrifuged at 14,000× g for 10 min. The fragmented DNAs in the supernatant were extracted and analyzed with 2% agarose gel electrophoresis containing 0.1 μg/mL ethidium bromide. The stained DNA fragments were imaged with Imagemaster VDS (Pharmacia, Uppsala, Sweden).

Cell cycle
Hep G2 cells were seeded at the density of 1.0 × 10 6 cells per 60-mm dish and treated with ACE polysaccharides (25 μg/mL), ACE/CS (ACE polysaccharides = 13.2 μg/mL) and ACE/S (ACE polysaccharides = 21.2 μg/mL) for 24 h and 48 h, and then were harvested and fixed in ice cold 70% ethanol (v/v). The cells were further washed with phosphate-buffered saline (pH 7.2), incubated with 25 μg/mL RNase A at 37°C for 15 min and stained with 50 μg/mL propidium iodide (PI) for 30 min in the dark. In the flow cytometry assay, a FACSCalibur Flow Cytometer (Becton Dickinson, BD Biosciences, CA, USA) with an excitation wavelength 488 nm and an emission wavelength 630 nm was used, and data were acquired using Cell Quest software based on a minimum of 10 5 cells per sample.

Mitochondrial membrane potential analysis
Again, the Hep G2 cells were treated with ACE polysaccharides (25 μg/mL), ACE/CS (ACE polysaccharides = 13.2 μg/mL) and ACE/S (ACE polysaccharides = 21.2 μg/mL) at 37°C for 48 h. After being resuspended at a density of 1.0 × 10 6 cells/mL in PBS, the cells were stained with 25 μM Rhodamine 123 for 30 min at 37°C and the membrane potential (ΔC) was detected by a FACSCalibur Flow Cytometer with excitation at 488 nm and emission at 520 nm based on a minimum of 10 5 cells per sample.

Measurement of intracellular reactive oxygen species
Formation of reactive oxygen species (ROS) in cells was evaluated according to the modified method adapted from Chou and Lin [27]. Briefly, Hep G2 cells were suspended in DMEM/ 10% FBS with or without ACE polysaccharides (25 μg/mL), ACE/CS (ACE polysaccharides = 13.2 μg/mL) and ACE/S (ACE polysaccharides = 21.2 μg/mL) at 37°C for 48 h. After trypsinization, the cells were washed, resuspended at a density of 1.0 × 10 6 cells/mL in PBS and then kept in a dark chamber containing DCFDA (20 μM) for further flow cytometric analysis (488 nm excitation/520 nm emission).

Assay for Fas (apoptosis antigen 1, APO-1/cluster of differentiation 95, CD 95)
Hep G2 cells were treated with or without ACE polysaccharides, ACE/CS and ACE/S in the same concentrations described above at 37°C for 48 h, trypsinized, washed, resuspended at a density of 1.0 × 10 6 cells/mL in PBS and incubated with phycoerythin (PE)-conjugated Fas monoclonal antibody UB2 (1 pg/mL) at 4°C for 1 h. Samples were then valuated by a FACSCalibur Flow Cytometer with excitation 488 nm/emission 580 nm and data were acquired using Cell Quest software based on a minimum of 10 5 cells per sample.

Activity of caspase-3, caspase-8 and caspase-9
Activities of caspase-3, caspase-8 and caspase-9 were determined with a CaspGLOW Fluorescein Active Caspase Staining Kit (BioVision, USA). In brief, cells were pre-incubated with ACE polysaccharides (25 μg/mL), ACE/CS (ACE polysaccharides = 13.2 μg/mL) and ACE/S (ACE polysaccharides = 21.2 μg/mL) for 48 h and counted to 1.0 × 10 6 cells/60-mm dish. After centrifuging at 400× g for 10 min, the cell pellets were lysed and kept on ice for 10 min. Supernatants were collected after centrifuging at 10,000× g at 4°C for 3 min and DEVD-pNA (Asp-Glu-Val-Asp p-nitroaniline, for caspase-3), IETD-pNA (Ile-Glu-Thr-Asp p-nitroaniline, for caspase-8), LEHD-pNA (Leu-Glu-His-Asp p-nitroaniline, for caspase-9) being added to a final concentration of 50 mM. Each sample was further incubated at 37°C for 1 h in a water bath and fluorescence was detected by a FACSCalibur Flow Cytometer with excitation at 485 nm and emission at 520 nm based on a minimum of 10 5 cells per sample.

Statistical analysis
All data from the experiments were expressed as the mean ± SD and shown with error bars. A one-way ANOVA followed by Duncan's test was used for significance testing, using a p value of 0.05 (SPSS 11, SPSS Inc., Chicago, IL).

ACE polysaccharides, ACE/CS and ACE/S induce Hep G2 death and LDH release
The death of Hep G2 cells was induced by ACE polysaccharides, ACE/CS and ACE/S in a dose and time-dependent manner (Fig 1). The results showed a slight decrease in cell viability at 24 h but became very significant after 48 h incubation, especially in the ACE/CS group (Fig 1b). Meanwhile, the viability of Hep G2 cells only decreased to approximately 85% in chitosan-silica nanoparticle (CSNP) or silica nanoparticle (SNP) treatments, which without ACE polysaccharides were loaded even at the highest concentration of 667 μg/mL for 48 h incubation (Fig 1c). Regarding morphology, cell shrinkage was obviously observed in phase-contract micrographs when exposed to ACE/CS (Fig 2a-2d) and a positive control (Fig 2e), but was insignificant in the CSNP treatment (Fig 2g). The effects of ACE polysaccharides and synthesized nanoparticles on Hep G2 cell membrane integrity, determined by a LDH assay, are shown in Fig 3a-3c. ACE polysaccharides, ACE/CS and ACE/S caused cell membrane damage in a dose-and timedependent manner, especially at the highest concentration of 166.67 μg/mL ACE polysaccharide treatments for 48 h (> 60% LDH released). At the corresponding concentration; e.g., 166.67 μg/mL, ACE/ES and ACE/S (containing 13.2 μg/mL and 21.2 μg/mL ACE polysaccharides, respectively) showed much less membrane damage (< 20% LDH released ; Fig 3b and  3c). The effects of nanoparticles without ACE polysaccharides on Hep G2 cell membrane integrity are shown in Fig 3d. None of the chitosan-silica composite treatments made a significant   membranolytic effect up to 48 h even at a concentration of 667 μg/mL (< 3.7% LDH released). In contrast, silica nanoparticles were found to cause slight (< 6.5% LDH released) membrane damage in the same condition.

ACE polysaccharides, ACE/CS and ACE/S caused DNA fragmentation and apoptosis
The characteristic DNA laddering of apoptosis was carried out in Hep G2 cells treated with ACE polysaccharides, ACE/CS and ACE/S at the concentrations of 25 μg/mL, 13.2 μg/mL and 21.2 μg/mL, respectively, by using agarose gel electrophoresis. The results showed a ladder-like pattern of multiple DNA fragments consisting of approximately 180-200 base pairs (Fig 4). In cell cycle analysis, flow cytometry for quantifying the DNA fragmentation extent was used and the apoptotic cells were identified as the hypodiploidy peak (sub-G 1 ) on the PI histogram. The percentage of apoptotic cells was 1.42% in the control group and reached 1.46% after treatment with ACE polysaccharides (25 μg/mL) in 24 h (Table 1 and S1 Fig). When cells were incubated with ACE/S (containing 21.2 μg/mL ACE polysaccharides) for 24 h, the sub-G 1 phase population slightly increased to 2.76%, whereas it increased to 2.88% for the cells incubated in ACE/ CS (containing 13.2 μg/mL ACE polysaccharides; Table 1 and S1 Fig). After 48 h incubation, the sub-G 1 phase population approached 5.18% in the control group and reached 20.98% or 20.56% in ACE polysaccharides or ACE/CS group, respectively, but only increased to 10.86% for cells incubated with ACE/S (Table 1 and S1 Fig). In contrast, after Hep G2 was incubated with silica and chitosan-silica nanoparticles without ACE polysaccharides even at the highest concentration (667 μg/mL) for 48 h, the sub-G 1 phase population was just slightly increased to 6.54% and 9.16% (Table 1).

ACE polysaccharides, ACE/CS and ACE/S decreased the ΔΨm
To investigate the Hep G2 cell mitochondrial apoptotic events induced by ACE polysaccharides, ACE/CS and ACE/S, we analyzed the values of the mitochondrial membrane potential (ΔCm). As shown in Table 2

ACE polysaccharides, ACE/CS and ACE/S increased the ROS generation
The DCF fluorescent response, which indicates the level of ROS, was increased significantly in Hep G2 cells after incubation with ACE polysaccharides, ACE/CS or ACE/S, as shown in Table 2 and S3 Fig. After 48 h of incubation, the ROS formation increased to 18.76% in the ACE polysaccharide treatment relative to controls and increased to 7.06% and 13.36% in ACE/ CS and ACE/S treatments, respectively. The effects of SNP or CSNP on ROS generation are Hep G2 cells were seeded at the density of 1.0 × 10 6 cells per 60-mm dish and treated with ACE polysaccharides (25 μg/mL), ACE/CS (ACE polysaccharides = 13.2 μg/mL) and ACE/S (ACE polysaccharides = 21.2 μg/mL). The nanoparticles without ACE polysaccharides (CSNP and SNP) were also examined. The cells without any treatment were defined as controls. Flow cytometry with excitation wavelength 488 nm/emission wavelength 630 nm was used for the analysis of PI-stained DNA, and data acquired from Cell Quest software based on a minimum of 10 5 cells per sample were analyzed using a one-way ANOVA followed by Duncan's test. The results are shown as the mean ± standard deviation (S.D.; n = 3), which without a common superscript ( a , b and c ) at the same row were significantly different (p<0.05 also shown in Table 2 and none of them caused significant ROS production (< 3.52%) in Hep G2 cells even at a concentration of 667 μg/mL for 48 h incubation.

ACE polysaccharides, ACE/CS and ACE/S increased the expressions of Fas/APO-1 and caspase activities
The Fas/FasL system is the most important apoptosis initiator in cells and tissues. In this study, the expression of Fas/APO-1 was detected in Hep G2 cells after treatment with ACE polysaccharides, ACE/CS and ACE/S. The Fas/APO-1 content increased to 47.6% relative to controls after incubation with ACE polysaccharides, ACE/CS or ACE/S (

Discussion
In our previous study, the major composition of ACE was polysaccharides with a (1!3)-β-Dglucan structure, while the average particle sizes and encapsulation efficiencies of ACE/CS and ACE/S were 210 ± 13.3 nm and 294 ± 25.7 nm and 85.7% and 76.4%, respectively [28]. Many functions of polysaccharides extracted from A. camphorate have been identified [3,10,11]. In this study, we revealed ACE polysaccharides, ACE/CS and ACE/S all showed interesting cytotoxicity activities against Hep G2 cells, i.e., antitumor activity, especially with the ACE/CS treatment. Therefore, we further examined the cell morphology by phase-contrast microscopy in the ACE/CS group and cell shrinkage was obviously present. Apparently, the nano-encapsulation of ACE polysaccharides provided a viable approach for enhancing antitumor efficacy in liver cancer cells. Moreover, previous studies showed that polysaccharides extracted from A. camphorata had no cytotoxic effect on human leukemic U937 cells, human endothelial cells and normal hepatocytes cells [11]. Our earlier study also reported ACE polysaccharides, ACE/ CS or ACE/S were nontoxic to three human normal cell lines; i.e., human skin fibroblast CCD-966sk cells, human normal fibroblast WS1 cells and human lung embryonic MRC-5 cells [28]. These studies suggested that AEC polysaccharides, ACE/CS and ACE/S are only toxic to cancer cells, such as Hep G2 cells, but not to normal cells. In contrast, the cytotoxicity of chitosan-silica or silica nanoparticles without ACE polysaccharides loaded on Hep G2 cells was insignificant, and this result was consistent with the results of our previous study, which demonstrated that silica nanoparticles became more toxic in normal human fibroblast cells than in cancer cells at high concentrations [25]. LDH leakage from cells is the explicit evidence of cell membrane damage and refers to one of the physiological features of necrotic cell death [29]. In the present study, we found LDH leakage was increased as the concentrations of ACE polysaccharides, ACE/CS and ACE/S treatments increased in the Hep G2 cells. These results were in agreement with the data obtained from the MTT assay (Fig 1). Meanwhile, 166.67 μg/mL of ACE/CS and ACE/S (containing 13.2 μg/mL and 21.2 μg/mL ACE polysaccharides, respectively) induced smaller membrane damage (< 20% LDH released) than the cytotoxicity (< 40% cell viability) released at the corresponding concentrations. The slight difference between cell viability and membrane damage could result from apoptosis and necrosis that existed concurrently when induced by nanoparticles [30,31]. However, at the highest concentration (166.67 μg/mL), ACE polysaccharides caused a massive (> 60% LDH released) membranolytic effect on Hep G2 cells in 48 h; hence, we deduced that high concentrations of ACE polysaccharides may induce cell necrosis. Therefore, the concentrations of ACE polysaccharides in the following tests were maintained at the level below the cytotoxic effect to evaluate the apoptosis mechanisms in Hep G2 cells.
Fragmentation of DNA is one of the most important and irreversible events in apoptosis. To further examine cell apoptosis that was not affected by necrosis, DNA fragmentation and a cell cycle assay were performed in this study. The results revealed that ACE polysaccharides, ACE/CS or ACE/S treatments could induce DNA degradation and resulted in the appearance of a ladder-like pattern. The cell cycle study also showed that the ACE polysaccharides, ACE/S and ACE/S increased the sub-G 1 phase population of Hep G2 cells in a time-dependent manner. Research has shown that A. camphorata can cause the accumulation of the sub-G 1 population with hypodiploidic DNA content, which is a typical marker of apoptosis and triggers cell death [8]. The assays described above demonstrated that the ACE polysaccharides and resulting nanoparticles were capable of inducing apoptosis and triggering cell death.
In previous studies, A. camphorata could induce apoptosis in cancer cells by triggering the mitochondrial pathway [5]. In this study, the ΔCm of Hep G2 cells decreased dramatically after 48 h of incubation with ACE polysaccharides, ACE/CS or ACE/S, which indicated that ACE polysaccharides and the resulting nanoparticles could induce the apoptosis of Hep G2 cells through damage of the mitochondrial membrane. Meanwhile, the factors of oxidative stress, such as ROS and lipid peroxidation, have been observed in some apoptotic processes [32][33][34]. ROS plays an important role in apoptosis by regulating the activity of certain enzymes involved in the cell death pathway. In our study, the incubation of Hep G2 cells with ACE polysaccharides, ACE/CS or ACE/S induced ROS generation and caused the subsequent apoptosis. However, a previous study revealed that A. camphorata might possess antioxidant properties against ROS generation [35]. This conflict showed the active components in A. camphorata might serve as mediators of the reactive oxygen scavenging system and have the potential to act as both pro-oxidants and antioxidants, depending on the redox state of the biological environment.
The Fas/FasL system is a key signaling transduction pathway of apoptosis in cells and tissues [36,37]. Many publications have highlighted the role of the Fas/FasL system in chemotherapyinduced apoptosis of tumors by up-regulating Fas/APO-1 or FasL [38]. In this study, we found Fas/APO-1 expression in Hep G2 cells dramatically increased after incubation with ACE polysaccharides, ACE/CS or ACE/S, meaning those materials have the ability to induce Fas-mediated apoptosis. In the case of caspases, the proteolytic activities of caspase-3, caspase-8, and caspase-9 on Hep G2 cells increased from 19% to nearly 42% after ACE polysaccharides, ACE/ CS or ACE/S incubation for 48 h. Activated caspase-8 and caspase-9 further initiated the activation of the caspase cascade leading to biochemical and morphological changes associated with apoptosis [39][40][41]. Caspase-3 is a well-known downstream effector of caspase that can be activated by caspase-8 or caspase-9 via different signaling pathways [42]. The results of our study demonstrated that ACE polysaccharides, ACE/CS and ACE/S activated the caspase cascade and resulted in Hep G2 cell death.
The results of the apoptosis assays shown above revealed that ACE polysaccharides, ACE/ CS or ACE/S induced similar apoptosis mechanisms in the Hep G2 cells, but it took a smaller dose of ACE/CS (ACE polysaccharides = 13.2 μg/mL) to reach the same effect than in ACE/S (ACE polysaccharides = 21.2 μg/mL) and ACE polysaccharides (25 μg/mL). This result suggested that nano-encapsulation increased the apoptosis effect of ACE polysaccharides in the Hep G2 cells, and ACE/CS could have more potential than ACE/S. In conclusion, ACE polysaccharides, ACE/CS and ACE/S appear to be promising antitumor agents for liver cancer cells. The encapsulation of ACE polysaccharides by chitosan-silica nanoparticles might provide a useful carrier for antitumor ingredients.