Research focus: SWNHs (single-walled carbon nanohorns) may be utilized to treat cancer as drug carriers based on their particular structure. But, the effect mechanism of the material itself on liver cells has not been investigated. Research methods used: To answer those questions, the roles of rude SWNHs on the biology functions of human liver normal and cancer cells were studied. Results/findings of the research: The results indicated that SWNHs suppressed growth, proliferation, and mitotic entry of human liver normal and cancer cells, and also pushed cell apoptosis, especially in cancer cells. We had found SWNHs in the lysosomes of L02 cells and in the nuclei of HepG2 cells. It indicated that individual spherical SWNH particles could enter into liver cells. Main conclusions and recommendations: Our results identified that it had different mechanisms between SWNHs and hepatoma cell lines or human normal cell lines. Advanced research about application and mechanisms in the treatment of HCC (hepatocellular carcinoma) by SWNHs should be carried out.
- SWNHs (single-walled carbon nanohorns)
- hepatocellular carcinoma
SWNHs (single-walled carbon nanohorns) were first synthesized by Iijima et al.  in 1999. On the basis of their special surface structures and large surface areas, SWNHs may be used in pharmacy and biomedicine. [2, 8] Some investigators  have applied oxidized SWNHs (SWNHox) as drug carriers with cisplatin for treatment of cancer. They found that SWNHs could slowly release cisplatin and inhibit effectively the proliferation of human lung cancer cells NCI-H460.
The results from their research also demonstrated that SWNHox itself could not suppress or promote the proliferation of human cancer cells as carrier material. In addition, up to now, the functions of unembellished SWNHs on cells were not very apparent.  Many researchers have studied the biological functions of graphene, fullerene, and carbon nanotubes (CNTs). [10, 32] They found that nanoparticles of carbon could enter into cells, even subcellular organelles such as lysosomes or nuclei. [17, 21, 23, 24] Carbon nanoparticles can induce apoptosis related to oxidative stress. [33, 36]
How SWNHs influence the functions of L02 (human normal liver cell lines) and HepG2 (human hepatoma cell lines) was unknown. Moreover, the differences role mechanisms between the material and normal liver cells or cancer liver cells had not been studied. To answer these questions, the direct effects of SWNHs on human hepatoma cell line HepG2 and human liver normal cell line L02 were investigated. We performed internalization in liver cell lines with SWNH particles. The extraordinarily different role induced by SWNHs on hepatoma cell lines and liver normal cell lines, will be useful for the treatment of HCC (hepatocellular carcinoma). To understand how studies on nanomaterial toxicology might relate to nanomedicine safety, it is important to consider the current regulatory framework (legally binding requirements and guidelines) in place for all medicine evaluations.
2. Characterization of material
By arc-discharge method, SWNHs was synthesized in air. It’s characterized by high-resolution transmission electron microscopy (HRTEM), Raman spectra, and thermogravimetry, as previously reported . SWNHs are dahlia-like structures and spherical aggregates; the diameters of SWNHs ranged from 60–100 nm. We determined other elemental contents of SWNHs with S4 Explorer X-ray fluorescence spectrometer. We detected the elemental contents of SWNHs with rapid N tube.
2.1. Preparation of dishes coated with SWNHs
SWNHs were suspended in ultrapure dialysis water from Milli-Q Integral Water 5 Purification System. SWNHs solution (10 µg/mL) were placed on PS (polystyrene) dishes and dried at 80°C in air for 4 h. Then these dishes were treated with ultraviolet irradiation for 1 h. The abbreviations SWNHs10, SWNHs20, SWNHs30, and SWNHs40 correspond to 0.21 µg/cm2, 0.42 µg/cm2, 0.64 µg/cm2, and 0.85µg/cm2 in each dish (60 mm), respectively.  Dataphysics OCA 40 Contact Angle Measuring System was used to determine the contact angles of 2 μl volume water droplets on the noncoated PS surfaces and the SWNHs40-coated surfaces at 20°C.
2.2. Cell culture
HepG2 and L02 cells were seeded onto noncoated PS dishes and dishes were coated with SWNHs10, SWNHs20, SWNHs30, SWNHs40, and then cultured with DMEM (Dulbecco’s Modified Eagle’s Medium supplemented), 1% penicillin-streptomycin solution, and 10% fetal bovine serum at 37°C in 5% CO2. 
2.3. Growth curve and morphology of liver cells cultured with SWNHs
HepG2 (3×105) and L02 (3×105) cells were seeded onto dishes, respectively. After being cultured for 48 h, these cells were subsequently visualized with optical microscope, based on the general protocol. These cells were observed and photos were acquired with a camera. At 48 h, cells were numbered with cell counter plate. The polynomial fitting about relationship between quantities (µg) of SWNHs (NH) and number of cells (CN) was carried out with using Origin 8 software.
2.4. Measurement of mitotic entry
These cells were synchronized with thymidine and cultured on dishes coated with SWNHs for 16 h. Then the cells were released into cell cycle with fresh medium. In the end, these cells were gathered or treated at different time points for subsequent specific analyses. DNA synthesis was measured with bromodeoxyuridine (BrdU) labeling in these cells. The BrdU-positive and the total number of cells were counted. Mitotic cells were evaluated by time-lapse videomicroscopy with Openlab software. 
2.5. Assessment of SWNHs
Elemental composition of SWNHs included 0.25% total metal content and 95.3% carbon, each metal less than 0.1%. [39, 41] Our results indicated that the surface area of BET was 631.55m2/g.  The results with regard to adsorptive isotherm plot of SWNHs showed that the surface of SWNHs particles was hydrophobic.
At the relative pressure 0.994 (P/Po), the diameter of SWNHs material was less than 308.7 nm. Moreover, the pore volume of SWNHs was 1.57 cm3/g. The average pore diameter of SWNHs was 9.97 nm. The particle density was 1.0077 g/cm3. The pore size distribution by Barrett-Joyner-Halenda (BJH) indicated that diameters of most mesopores were 2–8 nm in the SWNHs material. These results showed that SWNHs had many closed pores. The dimension range of suspended SWNHs particle was 295–615 nm in aqueous solution.
2.6. Characterization of dishes coated with SWNHs
Single particles of SWNHs on the surface of dishes were showed in images from scanning electron microscope (SEM); their diameters were 60–100 nm (Figure 1). The aggregates of secondary SWNH were dispersed and exhibited on individual particles on the surface of PS dishes. On surface of PS dishes, the contact angle of water droplet was 44.9°, less than the dried SWNHs-coated surface at 74.5°. The hydrophobicity of uncoated PS surface was lower than that of SWNHs40/PS surface.
2.7. Morphology of cells
The morphology of cells cultured for 48 h in SWNH-coated and uncoated dishes was observed by optical microscope. The images showed that the number of L02 (Figure 2 A–C) and HepG2 (Figure 2 D–F) decreased in a concentration-dependent manner. More spherical cells could be observed, which became smaller in size. That was remarkable in HepG2 cells. SWNHs had a much larger effect on liver cancer cells than normal as proliferation inhibitor factor.
2.8. SWNHs inhibit mitotic entry, proliferation, and growth of liver cells
With increased quantities of SWNHs, the growth curves of HepG2 and L02 cells are shown at 48 h; both the amounts of L02 (Figure 2G, lower curve) and HepG2 (Figure 2H, upper curve) decreased noticeably in a concentration-dependent manner (
The polynomial fitting formula of HepG2 (upper dotted curve, Figure 2G) was as follows:
Both the relationships between the number of L02 or HepG2 cells and the quantities of SWNHs are quartic polynomial.
The first-order derivates of the above two formulas (d [CN]/d [NH]) were also deduced. The first-order derivate of L02 (upper curve, Figure 2H) was as follows:
The first-order derivate of HepG2 (lower curve, Figure 2H) was as follows:
First-order derivative denoted that the changed rate of cell numbers followed with the increasing concentration of SWNHs. The formula of the changed rate was negative value, which suggested that SWNHs suppressed proliferation of liver cells. Moreover, the low changed rate of HepG2 cells compared to L02 cells revealed the more obvious inhibitory effect of SWNHs on cancer cells than normal (Figure 2H).
Cells were mixed with BrdU to investigate the effect of SWNHs on mitosis entry. Our results indicated that mitotic entry of both cells was delayed noticeably by SWNHs (Figure 3A and B) and that proliferation of cells was suppressed (Figure 3C,D) in a concentration- and time-dependent manner (
The results of XTT assay suggested that HepG2 and L02 cells (Figure 4) all were noticeably inhibited in a concentration- and time-dependent manner (
2.9. Cell cycle affected by SWNHs
Cell cycle affected by SWNHS was detected with flow cytometry. Our results identified that cell cycles of L02 and HepG2 were affected by SWNHs (Figure 5), and in a concentration-dependent manner. The S phase of liver cells noticeably decreased, and the G1 phase increased obviously in a concentration-dependent manner (L02,
2.10. SWNHs induced apoptosis
The apoptotic cells were determined by 7-AAD and Annexin-V. Our results indicated that apoptosis of HepG2 and L02 (Figure 6) were caused by SWNHs in a concentration-dependent manner, especially in HepG2 (
Biochemical events of apoptosis induce characteristic changes and death of cells. These changes include chromosomal DNA fragmentation, chromatin condensation, nuclear fragmentation, cell shrinkage and blebbing. Apoptosis in mammalian animal cells is regulated by SIRT1 and p53. Caspases are a group of cysteine proteases, and the key factors in cell apoptosis, such as caspase-3 and caspase-7. Our results identified that the expression of p53, caspase-7, and caspase-3 increased in L02 and HepG2 (Figure 6) in a concentration-dependent manner. Moreover, activation cleavage SIRT1 expression decreased in a dose-dependent manner. It confirmed that apoptosis of liver cells was promoted by SWNHs.
2.11. Cellular morphology observed by confocal microscope
The confocal images showed the morphology of L02; cell shapes of their nuclei were similar to control (Figure 7A), and number of cells decreased in a concentration-dependent manner. In L02 treated with SWNHs, the size of nuclei was slightly larger than control (Figure 7B,C); the nuclei swelled following the increasing of SWNHs concentration.
The number of viable HepG2 decreased remarkably and apoptotic HepG2 increased in a concentration-dependent manner. Apoptotic HepG2 cells could be found in Figure 7F (showed as arrow), but could not be observed in controls (Figure 7D).
The images of HepG2 treated with SWNHs40 indicated that the size of nuclei was much smaller than control. As reported by Romero et al.,  karyopyknosis was induced by SWNHs (Figure 7 F, showed as arrows). The karyopyknosis and larger nuclei caused by SWNHs were benefit of increased protein content of earlier apoptosis and decreased content of late apoptosis in nuclei. It may elucidate the suddenly increased G2 phase in HepG2 cells deal with SWNHs 40 (Figure5B). HepG2 cells deal with SWNHs 40 was nearly all apoptotic, meanwhile the ratio of S phase decreased remarkably and cells were obstructed at the G2/M phase. But, few L02 cells were apoptosis effects induced by SWNHs. HepG2 deal with SWNHs40 illustrated classic apoptotic morphology, but there were not so obvious apoptotic L02 cells. The apoptotic cells showed typical features, such as cell shrinkage, chromatin condensation, membrane blebbing, scant cytoplasm, and apoptotic body, [43, 45] especially in HepG2 cells.
Cells deal with SWNHs40 (0.85 µg/cm2) were collected for Transmission Electron Microscopy (TEM) measurement. By TEM, SWNHs can be found in cells. In L02, SWNHs aggregate localized at lysosomes and was smaller than 100 nm (Figure 8B and C). Moreover, it localized at nuclei in HepG2 cells (Figure 8E and F). The individual particles of SWNHs were easily dispersed from their neighbor molecules based on SWNHs possess high electronic densities on the surface than other molecules in organelles. The TEM images of organelles showed that both lysosomes and mitochondria in L02 (Figure8G) and HepG2 (Figure8H) deal with SWNHs were larger than control, respectively. And it was more remarkable in HepG2. Outside L02 (Figure8G) and HepG2 cells (Figure8H) deal with SWNHs, there were a lot of secretory vesicles, but not seen outside control cells. The secretory vesicles outside HepG2 (Figure8H) deal with SWNHs were larger than those outside L02 (Figure8G). SWNHs were heterogeneous materials, so cells secreted ingested SWNHs as normal response. HepG2 may have ingested much more SWNHs than L02. And the more cells ingested SWNHs, the more there were secretory vesicles.
The aforementioned studies have illustrated that some carbon nanomaterials suppressed growth and proliferation of L02 or HepG2. Fullerene C60 inhibited proliferation of HepG2 at a low dosage of 0.46 µg/mL. Liu et al. suggested that unmodified MWNTs (multiwalled nanotubes), MWNTs modified with carboxyl, and MWNTs modified with hydroxyl at dosages of 12.5–200.0 µg/mL inhibited viability of L02 cells in a dose-dependent manner. Romero et al.  illustrated that poly [sulfopropyl methacrylate] (PSPM), or oxidized, lipid, poly [allylamine hydrochloride] (PAH)-modified MWNTs at higher dosage (100 µg/mL) suppressed growth and proliferation of HepG2. It was well known that SWCNTs (single-walled carbon nanotubes) had great potential to benefit biomedicine, for example, photothermal ablation therapy for tumor bond with near-infrared irradiation. Hashida et al.  reported the photothermal activity of a novel SWCNT bond with (KFKA) 7 peptide [H-(-Lys-Phe-Lys-Ala-)7-OH] with repeated structure, which could be used to treat tumor. The SWCNTs–(KFKA) 7 compounds could be used to ablate tumor in photothermal cancer therapy with its high aqueous dispersibility.  Our results were similar in HepG2 and L02 cells deal with SWNHs at low dose (1.5–6.0 µg/mL). Ajima et al.  reported that SWNHox (oxidized SWNHs) did not suppress or promote proliferation of lung cancer cells at dose of 0–20 µg/mL. But
After exposure to a chemical toxicant, the most easily and first visualized change and effect in cells could be observed come from cell morphology. So, morphological changes were benefit for toxicity biomarker. TEM could be used to identify the location of nanoparticles in organelles of cells based on its powerful denote to observing micron and nanometer structures. Oxidized SWNTs or modified and unmodified MWNTs could be found in endosomes, cytoplasm, or lysosomes of human macrophage cells by TEM. [19, 20] Porter et al.  found that dispersed SWNTs in tetrahydrofuran localized in nuclei and lysosomes of human macrophages. And fullerene C60 could be found in nuclei and cytoplasm of human macrophages.  Besides in endosomes, cytoplasm, or lysosomes, in human embryonic kidney 293 cells, MWNT-NH2 was first found in nuclei.  In HepG2 cells, the PSPM, lipid, or oxidized-modified MWNTs could be discovered in cytoplasm. However, there were no references involved in carbon nanoparticles locating in the nuclei of cells. Although, in nuclei of HepG2 cells, changes of protein and DNA had been obtained based on confocal Raman microscope induced by modified MWNTs.  Combining the results from TEM and confocal microscope, we identified the different interactive mechanisms between SWNHs and L02 or HepG2 cells, respectively. SWNHs could directly enter into nuclei of HepG2 and could induce cell apoptosis, while SWNHs could induce apoptosis of L02 cells associated with activation of lysosomal function.
Changes in growth of cells also could be determined as a toxic effect. The chemical toxicities could be assessed by an assay called plating efficiency. This evaluated the ability of cells to form colonies after 15 days of culture in the presence of a substance, signaling cell survival and ability to reproduce. Another assay that evaluated the capability of cells to replicate and determined the concentration of the substance at which 50% of the cells do not multiply is named the median inhibitory dose (ID50). Romero et al.  illustrated the effects of inhibiting role on cell proliferation caused by apoptosis, which as toxicity of carbon nanotubes (CNTs) for HepG2
In fact, SWNHs themselves suppressed growth and proliferation of HepG2 cells; meanwhile induced their apoptosis. It revealed that SWNHs material could be used as a drug carrier for anti-HCC, and carried anticancer drugs such as cisplatin based on its cooperative therapeutic role.
Nanoparticle almost all located in lysosome and was degradated and sequestrated. The mitochondrial apoptosis and lysosomal dysfunctions could induce toxicological consequences. [51, 52] Based on oxidative stress pathway related to apoptosis caused by nanoparticle,  localization of nanoparticles may induce activation of lysosome, which with high content of digestive enzyme, triggering mitochondrial apoptosis.  Mitochondria was the main basis of redox and ROS (reactive oxygen species) machinery in cells, and could activate inflammatory cytokines.  Oxidative stress toke place in HepG2 cells deal with oxidized or unmodified MWNTs and SWNTs, [25, 28] and in L02 cells deal with hydroxyl-modified, carboxyl-modified or unmodified MWNTs,  and in rat hepatocytes deal with fullerenes C60(OH)24, C60(OH)12, and C60.  Gao et al.  found that modified MWNTs internalized into human macrophages cells THP-1 and located in lysosomes, induced ROG generation and low mitochondrial membrane potential. Tahara et al.  identified that high concentration of SWNHs caused destabilization of lysosomal membrane and ROS generation in murine macrophage RAW 264.7. The results of us demonstrated individual SWNH particles located at mitochondria of L02 and HepG2, or lysosomes of L02 cells. It suggested that mitochondrial apoptosis and lysosomal dysfunctions in liver cells play key role on physiological process related to SWNHs exposure.
The model of transmembrane pathways divided MWNTs into two classes types, singles and clusters. The clusters of CNT were collected in cells associated with energy-dependent endocytosis process; meanwhile extremely dispersed single CNTs directly penetrated cellular membranes entried into cells. [2, 1] We will assure the transmembrane pathway of SWNHs. Because one spherical SWNH aggregate were assembled by about 2000 nanohorns, the aggregates showed clusters of SWNHs.  Nevertheless, the aggregate more like one single nanoparticle, and not easy to separate into individual nanohorns. Porter et al. [1, 8] identified the internalization of SWNTs with smaller diameters of SWNTs (0.6–3.5 nm) in cells through nuclear pore complex and other pores.  A single SWNHs aggregate has larger diameter (60–100 nm) than fullerene C60 (0.7 nm) and single SWNTs (0.6–3.5 nm). It is very interesting how aggregates of individual spherical SWNH composed with 2000 nanohorns internalize in cells and localize at lysosomes in L02 cells, and even entry into the nuclei of HepG2 cells. Because it was suggested that only nanoparticles less than 40 nm in diameter could enter into nuclei of cells.  The low negative charge density on surfaces of SWNHs and the bovine serum proteins used in cell culture may be benefit for SWNH dispersion in culture media, or induce SWNHs recognized with cellular membranes by receptors on the surface of cells. 
Researches in this area remain many questions. How SWNHs enter into different cells and locate at different destinations (such as nuclei, liposome, or mitochondria) in different cells? How SWNHs induce the apoptosis in different cells? However, toxicity is a complex event showing several effects, even cell death until metabolic aberrations. Neuro-, liver, and/or kidney toxicity are examples of functional changes not necessarily events linked to cell death. Due to these evidences, the
Nanotoxicology examines the bioeffects of nanomaterials concerning the toxic activities and depends on the
The cell culture assays have as advantages the fast results in the research, reduced cost, and a few quantities of substance to be used. Besides that, those models can predict toxicity in animals including humans to get more precise results to establish multitiered
In this way, the primary goal of