Nano Research DOI 10.1007/s12274-017-1590-7
Construction of highly stable selenium nanoparticles embedded in hollow nanofibers of polysaccharide and their antitumor activities Zhaohua Ping1, Ting Liu2, Hui Xu1, Yan Meng1, Wenhua Li2, Xiaojuan Xu1 (), and Lina Zhang1 () 1 2
College of Chemistry & Molecular Sciences, Wuhan University, Wuhan 430072, China Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan 430072, China
Received: 21 October 2016
ABSTRACT
Revised: 11 March 2017
Nanotechnologies have been exploited to develop safe and effective medicines and pharmaceuticals. In the present study, a novel functional nanomedicine constructed from a bioactive polysaccharide and selenium nanoparticles (SeNPs) was developed. A highly-branched β-(1→3)-D-glucan (AF1) with high anti-tumor activity was used to self-assemble hollow nanofibers with an apparent average diameter of 92 nm; Se nanoparticles were synthesized via the reduction of sodium selenite. The results of light scattering, transmission electron microscopy, and X-ray diffraction demonstrated that the spherical SeNPs with a mean diameter of 46 nm were entrapped in the cavities of the AF1 hollow nanofibers through the formation of Se–O bonds between SeNPs and AF1, leading to the good dispersion and high stability in water for over 16 months. In vitro and in vivo assays indicated that the AF1-Se nanocomposite had higher anti-tumor activities against breast cancer. Furthermore, AF1-Se displayed a broad-spectrum inhibition against human cancers with low half maximal inhibitory concentration (IC50) values and low toxicity to normal cells. Particularly, the inhibition ratio of AF1-Se against MCF-7 cancer cells reached 75% at a concentration of 200 μg·mL–1 with 29 μM Se content, much higher than that by treatment with AF1 alone, suggesting a strong synergic effect and nano impact. Overall, we developed a method for increasing the stability, anti-tumor activity, and safety of SeNPs by wrapping with bioactive polysaccharides.
Accepted: 13 March 2017 © Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2017
KEYWORDS selenium nanoparticles, stability and dispersion, bioactive polysaccharide, anti-cancer activity, synergistic effect
1
Introduction
The application of nanotechnologies in the medical field has revolutionized the landscape of the
pharmaceutical and biotechnology industries [1–3]. Designing multifunctional nanotherapeutic agents to improve patient outcomes is the goal of nanomedicine translation to the clinic [4, 5]. Cancer is a serious disease
Address correspondence to Xiaojuan Xu,
[email protected]; Lina Zhang,
[email protected]
2
Nano Res.
and great threat to public health; nearly 600,000 people with tumors in the USA died in 2016 [6]. Currently, the application of nanomaterials in biomedicine is increasing rapidly and offers excellent prospects for the development of new non-invasive strategies for the targeted therapy, molecular diagnosis, and treatment of cancer [7–9]. The use of nanoparticles as delivery vehicles for anticancer drugs has many unique advantages, including targeting action, reducing toxicity, and protection of drugs against degradation [10, 11]. However, a major challenge in fabricating nano-sized drug carriers is controlling the size, stability, and surface properties of nanoparticles, which determines the efficiency of this approach [12]. Selenium (Se) is not only an essential trace element in mammals, but also plays an important role in disease resistance, antitumor, and immunomodulating activities [13]. It is well-known that Se has a very narrow margin between its lowest acceptable levels of intake and toxicity, and selenium nanoparticles (SeNPs) can reduce the risk of Se toxicity compared to Se element alone [14, 15]. SeNPs have gained attention because of their facile functionalization chemistries, high anti-cancer activity, and fair biocompatibility [16–18]. However, SeNPs are also prone to aggregation into large clusters in aqueous solution, leading to lower bioactivity and bioavailability. Thus, much effort has been devoted to developing “green” methods for the synthesis and dispersion of SeNPs. Hu et al. reported that Se compounds can enhance paclitaxel drug efficacy against human prostate cancer and down-regulate anti-apoptotic proteins of Bcl-XL and survivin [19]. Yu et al. decorated SeNPs with folatechitosan to form FAC@CurP-SeNPs that can inhibit the growth of MCF-7 cells by inducing apoptosis [20]. Moreover, transferrin (Tf)-conjugated SeNPs loaded with doxorubicin strongly enhanced anti-cancer effects through apoptosis induced by both intrinsic and extrinsic pathways [21], and Tf-SeNPs significantly inhibited in vivo tumor growth in a nude mice model. Thus, selenocompounds are highly effective modulators of the therapeutic efficacy and selectivity of anti-cancer drugs. Because of the instability of SeNPs, however, searching for a suitable carrier of SeNPs is the key for their successful application in biomedicine. Reported carriers of SeNPs are typically modified before use,
leading to relatively complex preparation processes and difficulty in clinical translation. For a long time, some active polysaccharides have been used as cancer therapeutics to improve treatment outcomes with few side effects and significantly prolong the life span of cancer patients [22–24]. The well-known polysaccharide K and schizophyllan are currently used clinically as anti-tumor agents [25, 26] in Japan. Therefore, using bioactive polysaccharides to fabricate selenocompounds via a “green” route is a better strategy for effectively utilizing Se compared to synthetic polymers [27, 28]. In our laboratory, a highly-branched polysaccharide (AF1) with the β(1→3)-D-glucan as the backbone from fruiting bodies of Auricularia auricular-judae were found to exhibit significant anti-hepatoma activity without cytotoxicity [29, 30]. AF1 adopts a stiff chain conformation in water and shows strong self-assembly ability to form hollow fibers [31]. In the present study, glucan AF1 was used to wrap the SeNPs to improve the stability, safety, and antitumor activities of SeNPs as an alternate strategy. Here, facile synthesis of highly uniform SeNPs was discovered using AF1 glucan as a dispersant and stabilizer. The nano-structure and sizes of SeNPs and AF1-Se composites were studied by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS), and their application potential in biomedicine was evaluated. Thus, a novel strategy to disperse and stabilize SeNPs using a water-soluble polysaccharide via one step was provided, opening a new pathway for constructing SeNPs with high stability and dispersion for application in the nanomedical field.
2 2.1
Experimental section Materials
The AF1 glucan sample was isolated from fruiting bodies of A. auricular-judae, a commercial product cultivated in Fangxian (Hubei, China), according to our previously reported method [29]. The weightaverage molecular weight (Mw) of AF1 in water was determined to be 2.29 × 106 by laser light scattering [30]. Na2SeO3 and ascorbic acid were of analytical grade and purchased from Sinopharm Chemical Reagent
| www.editorialmanager.com/nare/default.asp
3
Nano Res.
Co., Ltd. (Shanghai, China). Thiazolyl blue tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), coumarin6, propidium iodide (PI), 2’,7’-dichlorofluorescein diacetate (DCF-DA), 4’,6-diamidino-2-phenyindole (DAPI), lyso-tracker DND-99, Hoechst 33342, and bicinchoninic acid (BCA) kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). The terminal transferase dUTP nick end labeling (TUNEL) assay kit was obtained from Roche Applied Science (Basel, Switzerland). Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose, Hycolon) and Roswell Park Memorial Institute (RPMI) 1640 medium, fetal calf serum, penicillin, trypsin and streptomycin (cell culturegrade) were purchased from Gibco (Grand Island, NY, USA). Horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit and goat anti-mouse) were purchased from Beyotime (Nantong, China). Antibodies were obtained from the following sources: Bcl-2 from Abcam; caspase-3 and caspase-9 from Cell Signaling Technology (Danvers, MA, USA); Poly(ADPribose) polymerase (PARP1) antibody from Abclone (Victoria, Australia); PdCD4 and actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other reagents used were of analytical grade. The water used in all experiments was Milli-Q water. 2.2
Preparation of SeNPs
SeNPs were prepared according to the following procedure. The reaction of selenious acid with ascorbic acid (Vc) was carried out as described by Li et al [32]. Briefly, a stock solution of 0.1 M Na2SeO3 was prepared by dissolving 172.94 mg of Na2SeO3 in a 10-mL volumetric flask, and then Vc aqueous solution (0.2 M) was freshly prepared before use. The freshly prepared AF1 (1 mg·mL−1) aqueous solution (100 mL) was mixed with 1 mL Na2SeO3 followed by continuously stirring for 10 min at 25 °C. Two milliliters of aqueous Vc solution was added dropwise into the resulting mixture and stirred for 24 h at 25 °C. The reacted products were dialyzed by using regenerated cellulose tubes (Mw cutoff 8,000) against Milli-Q water for 3 days. The resulting solution was finally lyophilized to obtain SeNPs/AF1 composites, coded as AF1-Se. Se content was determined by inductively couple plasma mass spectrometry (ICP-MS) analysis.
To quantify the in vitro cellular uptake of AF1-Se, a fluorescein dye coumarin-6 at a final concentration of 4 μg·mL−1 was added to the reaction system before mixing AF1 with Vc as previously described [21]. After reacting for 24 h at 25 °C, the solution was dialyzed against Milli-Q water until no Se and coumarin-6 were detected in the Milli-Q water outside of the dialysis bag by ICP-MS analysis and fluorescence spectrophotometry, respectively. The incorporated coumarin-6 acted as a probe for AF1-Se and offered a sensitive method for determining its intracellular uptake and localization. 2.3 Characterization The AF1-Se nanocomposite was characterized by different microscopic and spectroscopic measurements. Fourier-transform infrared (FT-IR) spectra of the samples were recorded on a PerkinElmer FT-IR spectrometer (Nicolet 5700, PerkinElmer, Waltham, MA, USA) in the range of 4,000–400 cm−1 using the KBr-disk method. SEM was conducted by field emission scanning electron microscopy (FESEM, Zeiss, Jena, Germany) using an accelerating voltage of 5 kV. The samples were sputtered with gold before observation. SEM-EDX analysis was carried out on an EX-250 system (Horiba, Edison, NJ, USA) and employed to examine the elemental composition of AF1-Se powder. TEM (JEM-2010HR, Jeol, Tokyo, Japan) and high-resolution transmission electron microscopy (HRTEM, JEM2010FEF) were conducted at 200 kV. Wide-angle X-ray diffraction (WAXD) measurements of SeNPs, AF1, and AF1-Se were carried out on a WAXD diffractometer (D8-Advance, Bruker, Billerica, MA, USA). A commercial light-scattering spectrometer (ALV/CGS-3, ALV, Langen, Germany) equipped with an ALV-5000/ EPP multi-τ digital time correlator and He–Ne laser (at λ = 632.8 nm) was used at a scattering angle θ of 90°. All diluted solutions were optically cleaned by filtration through 0.45-μm Millipore filters (NYL, 13-mm syringe filter, Whatman, Inc., Maidstone, UK). Zeta potential and size distribution of the nanoparticles were measured using a Nano-ZS ZEN3600 (Malvern Instruments, Malvern, UK). X-ray photoelectron spectra (XPS) were recorded on a Kratos XSAM800 X-ray photoelectron spectrometer (Manchester, UK) using Mg Kα radiation as an excitation source.
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
4 2.4
Nano Res.
In vitro bioactivity assay
Human breast adenocarcinoma cell lines (MCF-7, MDA-MB-231, and MDA-MB-468), human hepatocellular carcinoma cell lines (BEL7402, Huh-7, HCCLM9, HepG2, and Hep3B), and normal cell lines (HBL-100, L02, and 293T) were purchased from the China Center for Type Culture Collection (Wuhan, China). HCCLM9 and L02 cells were cultured in RPMI 1640 medium, while the others were cultured in complete DMEM medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U·mL−1), and streptomycin (100 μg·mL−1). Cells were all cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The cytotoxicity of AF1-Se was evaluated by MTT assay. Cellular uptake by MCF-7 cells was quantitatively estimated by the fluorescence intensity of cell lysates by using a microplate reader (TECAN, SPARK10M, Männedorf, Switzerland) and laser-scanning confocal microscope (Nikon C1-Si TE 2000, Tokyo, Japan). The cell apoptosis, cell cycle distribution and intracellular reactive oxygen species (ROS) were analyzed by flow cytometry (Beckman, Brea, CA, USA). Data were processed using FlowJo software (Tree Star, Ashland, OR, USA). AF1-Se-induced DNA fragmentation in apoptotic cells was detected using a TUNEL assay following the manufacturer’s protocol observed under a fluorescence microscope (NIKON ECLIPSE TI-SR). 2.5
MCF-7 tumor xenograft assay
Animal experiments were conducted according to the guidelines of the Laboratory Animal Center of the Wuhan University College of Life Sciences. Fiveweek-old female athymic nude mice (BALB/c, nu/nu) were purchased from the Model Animal Research Center (Changsha, China). All experiments were conducted under approved procedures. All qualified mice were injected in the right armpit with 5 × 106 MCF-7 cells suspended in 0.2 mL of phosphate buffered saline (PBS). Three weeks later, tumor lumps had reached approximately 50 mm3, and tumor-bearing mice were randomly grouped into four experimental categories: treated daily with intraperitoneally injection of PBS (negative control), AF1 (5 mg·kg−1), and AF1-Se (5 mg·kg−1, 10 mg·kg−1) for 21 days. The tumor sizes and body weights of mice were measured every 2 days.
Tumor volumes were calculated as length × width2/2 [33]. After treatment for 21 days, the nude mice were sacrificed and the tumors and organs were excised, photographed, and weighed. 2.6 Western blot analysis Cellular and tumor tissues proteins were prepared as previously described, and western blot analysis was carried out as described in our previous work [30, 34]. 2.7
Statistical analysis
All experiments were performed at least in triplicate and the results were expressed as the mean ± SD. Statistical analysis was performed using SPSS version 19 software (SPSS Inc., Chicago, IL, USA). The differences between the control and tested groups were analyzed by Student’s t-test. Differences with *p < 0.05 or **p < 0.001 were considered statistically significant.
3
Result and discussion
3.1 Construction and morphology of SeNPs To improve the poor stability of SeNPs in water, the AF1 polysaccharide was used to wrap SeNPs. As shown in Scheme 1, the AF1 glucan extracted from the fruiting bodies of A. auricula-judae was dissolved in water and then self-assembled into hollow nanofibers, similarly to that in our previous work [31]. The AF1 hollow nanofibers helped to disperse and stabilize the SeNPs. To wrap SeNPs into the cavity of AF1 hollow nanofibers, selenious acids were first mixed well in aqueous AF1 solution (1 mg·mL–1), followed by the dropwise addition of ascorbic acid solution into the system. Accordingly, ascorbic acid reacted with selenious acid, resulting in the formation of SeNPs, supported by visible color changes. The solution color changed gradually from colorless to pale yellow and then to yellow in the presence of AF1, and AF1 nanofibers containing SeNPs were coded as AF1-Se. Figure 1 shows the structure and morphology of AF1 and AF1-Se. AF1 self-assembled to form hollow fibers with an apparent average diameter of 92 nm measured from TEM images, whereas the diameter of the AF1-Se nanofibers was approximately 193 nm,
| www.editorialmanager.com/nare/default.asp
5
Nano Res.
Scheme 1 Schematic illustration of preparation of AF1-Se nanocomposite.
Figure 1 Characterization of AF1-Se. (a) TEM image of nanofibers formed by AF1 in water at a concentration of 1 mg·mL−1. (b) and (c) TEM images of selenium nanoparticles dispersed in water with the AF1 concentration of 1 mg·mL−1. (d) Size distribution of SeNPs in (b). (e) SEM-EDX analysis of AF1-Se. (f) Selected area electron diffraction (SAED) image of Se in (b).
which was much larger than that of AF1, as a result of wrapping SeNPs. The long length of AF1-Se of several micrometers was ascribed to further aggregation during drying before SEM and TEM observation; however, this was not the true length of AF1-Se in solution. Because of the easy aggregation of AF1, the size strongly depends on the sample preparation. SEM images (Fig. S2 in the Electronic Supplementary Material (ESM)) of AF1-Se indicated that the AF1-Se nanofibers had uniform diameters of approximately 600 nm and continuous lengths. For TEM observation, the nanofibers were prepared by drying a small amount of dilute solutions on a copper grid, leading to the formation of the long nanofibers via “head to tail” linkage of a few AF1 chains during drying; in contrast, the nanofibers for SEM were prepared from a relatively large amount of solutions and frozen in liquid nitrogen and then lyophilized, leading to further aggregation of the nanofibers in parallel and “head to tail” linkage through hydrogen bonding, resulting in the formation of the longer and larger nanofibers than those in TEM [35]. Thus, the lengths of the AF1 and AF1-Se samples measured by TEM and SEM differed from those in solution. According to our previous work, the average contour length (L) of AF1 in water was approximately 1,300 nm [30], and AF1-Se at the same concentration should be similar to bare AF1. Furthermore, the data were comparable
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
6
Nano Res.
with those estimated by CONTIN analysis of DLS measurements (Fig. 3(c)). As shown in Figs. 1(b) and 1(c), SeNPs adopted a homogeneous spherical structure and were embedded in the AF1 tubes through physical interactions. The statistical average diameter of the SeNPs in the AF1Se system was estimated to be approximately 46 nm, as shown in Fig. 1(d). EDX analysis of the elemental composition of the AF1-Se indicated that carbon, oxygen, and selenium constituted the composite with 57.6%, 35.0%, and 7.4%, respectively (Fig. 1(e)), suggesting that the Se nanoparticles were successfully synthesized in the presence of AF1. The TEM images of AF1-Se (Figs. 1(b) and 1(c)) indicated that SeNPs were uniformly dispersed in the AF1 nanotubes, but not on their surface. From HR-TEM images (Fig. 1(f)), the electron diffraction pattern of AF1-Se was ambiguous compared with the clearly distinguished diffraction rings of pure SeNPs (Fig. S1 in the ESM), which was attributed to the shielding effect from AF1 hollow nanofibers. Importantly, SeNPs were not observed on the surface of AF1 nanofibers (Fig. S2 in the ESM), suggesting that SeNPs were mainly entrapped in the cavity of the hollow nanofibers. The zeta potential difference between the pure SeNPs (−22.8 mV) and AF1-Se (−3.28 mV) (Fig. 2(d)) further confirmed that SeNPs were shielded by AF1 nanofibers. The SeNPs were entrapped in the cavities of AF1 hollow nanofibers, shielding the partial negative charges of SeNPs and leading to smaller negative potentials. Furthermore, Fig. 2(a) shows strong and sharp peaks at 24° and 30°, corresponding to the (100) and (101) lattice planes of SeNPs [36]. These results demonstrate that pure SeNPs were successfully produced in this redox system. However, the two peaks at 24° and 30° completely disappeared in the presence of AF1 and showed the same pattern as that of the amorphous AF1. Peaks at 24° and 30° of SeNPs reappeared after adding DMSO to the AF1-Se dispersion (coded as DMSO-AF1-Se), demonstrating that SeNPs were entrapped by AF1 nanofibers. These results strongly indicate that SeNPs were successfully synthesized and dispersed in the AF1 hollow nanofibers in the aqueous solution to form AF1-Se nanocomposites.
Figure 2 Chemical composition and structural characterization of AF1-Se. (a) XRD patterns of SeNPs, AF1, AF1-Se, and DMSOAF1-Se. (b) XPS spectra of AF1 and AF1-Se. (c) O 1s spectra of AF1 and AF1-Se. (d) Zeta potentials of SeNPs (100 μM), AF1 (1 mg·mL−1), and AF1-Se (1 mg·mL−1).
Furthermore, SeNPs were mainly embedded in the trunk of AF1 hollow nanofibers with an apparent average diameter of 193 nm. Notably, bare SeNPs without wrapping with AF1 easily aggregated and precipitated because of their high surface energy (Fig. S3 in the ESM). Therefore, AF1 is an excellent disperser and can stabilize SeNPs in water for longterm storage. As reported previously, 5-fluorouracil can be conjugated to the surface of SeNPs; SeNPs were capped with 5-fluorouracil molecules to form compact and stable globular nanocomposites through Se–O/N bonds [16]. AF1 is a highly-branched biomacromolecule with a large number of hydroxyl groups and easily conjugates with the surface of SeNPs to form AF1-Se composites. To evaluate the interaction between AF1 and SeNPs, XPS spectra were recorded. As shown in Figs. 2(b) and 2(c), a change (∆V = 0.44 eV) in O 1s peaks between AF1 and AF1-Se was observed, which may correspond to the formation of an Se–O bond. It was speculated that some OH groups of the AF1 backbone were ligated with Se. It has been demonstrated that the AF1 backbone is located in the interior of the nanofibers, whereas side chains protruded outside the nanofibers because of their relatively hydrophilic nature [31]. Moreover, a blue shift was
| www.editorialmanager.com/nare/default.asp
7
Nano Res.
observed for the group of C–O at ~1,100 cm−1 in the FT-IR spectra of AF1-Se (Fig. S4 in the ESM), which may be ascribed to the formation of Se–O bonds [16]. Taken together, changes in the XPS and FT-IR spectra supported the formation of Se–O bonds in AF1-Se nanocomposites. 3.2
Stability of AF1-Se nanocomposites
The stability of Se nanomaterials is an important factor affecting its medicinal application [37, 38]. Figure 3(a) shows photographs of AF1-Se aqueous solution after storage for 1 day and 16 months. The AF1-Se solution displayed a yellow color because of the nanoscale effect of Se, and was quite stable, transparent, and lucent without any discernible precipitation after at least 16 months of storage, indicating excellent stability. The Z-average particle sizes of AF1-Se in both aqueous and physiological conditions (PBS, pH 7.4) determined by a particle size analyzer revealed that the AF1-Se solution remained stable within the examined time range of 0–240 h (Fig. 3(b)). Additionally, DLS is a powerful technique for analyzing particle size and the size distribution of the nanoscale particles in solution [39, 40]. CONTIN analysis of DLS measurements on
Figure 3 Stability of AF1-Se nanocomposites. (a) Solution color of AF1-Se aqueous solution (1 mg·mL−1) at room temperature after storage for 1 day and 16 months. (b) Stability of AF1-Se in aqueous and PBS (pH 7.4) solutions. (c) Rh distribution of AF1 and AF1-Se in aqueous solution with AF1 concentration of 1 mg·mL−1 at θ = 90° and T = 25 °C.
the AF1 and AF1-Se aqueous solution was performed; the results are shown in Fig. 3(c). The peaks with lower hydrodynamic radii (Rh) represented individual AF1 chains, whereas those with higher Rh values were attributed to their aggregates. The stiff AF1 chains could easily self-assemble and align in parallel into nanofibers with relatively hydrophobic hollow structures, driven by hydrogen bonding and hydrophobic interactions [31]. The results further confirmed that AF1-Se nanofibers formed in the AF1 solution, and SeNPs mainly embedded the inner cavity of the AF1 nanotubes. All of these results confirm that SeNPs were well-dispersed and stabilized by AF1 by forming nanocomposites of AF1-Se. AF1-Se may be widely applicable as a nanomedical material. 3.3
Enhanced cellular uptake of AF1-SeNPs in vitro
Cellular uptake of nanomaterial-based drugs is very important for medicinal application. AF1-Se nanofibers with an average length of ~1,000 nm and Z-average particle sizes of ~200 nm in the solution state should be taken up by cells according to previous studies [41, 16]. The fluorescent dye 6-coumarin is often used to determine the in vitro cellular uptake of nanoparticles [32]. Thus, the coumarin-6-loaded AF1-SeNP composite and SeNPs were prepared and cellular uptake by human breast adenocarcinoma MCF-7 cells was quantitatively analyzed by measuring the fluorescence intensity from intracellular coumarin-6-loaded nanoparticles before the activity assay. As shown in Fig. 4(a), after 6-coumarin was loaded into AF1-Se and taken up by the cells, intracellular AF1-Se concentrations increased in a time- and dose-dependent manner and were much higher than the values for SeNPs. After 2 h incubation with 40 and 80 g·mL−1 6-coumarinloaded nanoparticles, the intracellular concentrations of AF1-Se increased to 19.36 and 28.68 g per 106 cells, respectively, which were approximately 4–5-fold of that of 800 g·mL−1 SeNPs without AF1. The lower cellular uptake of SeNPs may be ascribed to the larger size of the SeNP cluster formed through aggregation. At 4 °C, few AF1-Se samples were taken into the cells (Fig. 4(b)), suggesting that AF1-Se was taken up through energy-dependent endocytosis, an important entry mechanism for extracellular materials, particularly
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
8
Nano Res.
Figure 4 In vitro cellular uptake and localization of AF1-Se. (a) and (b) Quantitative analysis of cellular uptake efficiency of 6-coumarinloaded AF1-Se and SeNPs at 37 °C or 6-coumarin-loaded AF1-Se at 4 °C by using a microplate reader to detect fluorescence intensity of MCF-7 cell lysates after 10 min, 1 h, and 2 h incubation, respectively. The data represent the average of at least three independent experiments ± SD. (c) MCF-7 cells were stained with 100 nM of lyso-tracker DND-99 (red fluorescence) for 30 min and 10 μg·mL−1 of Hoechst 33342 (blue fluorescence) for 20 min, and then treated with 50 μg·mL−1 6-coumarin-loaded nanoparticles (green fluorescence) for different time, followed by visualization under a fluorescence microscope.
nanomaterials [42]. Therefore, AF1 significantly enhanced the cellular uptake of SeNPs. Fluorescence imaging techniques provided insight into the intracellular trafficking of AF1-Se. As shown in Fig. 4(c), blue-labeled cell nuclei were circumvented by green fluorescence indicating 6-coumarin loaded AF1-Se, and no overlay of green and blue fluorescence was observed. In contrast, the green and red fluorescence corresponding to lysosomes clearly overplayed after 10 min incubation, suggesting that lysosomes were the main target organelles of AF1-Se. To further confirm AF1 was taken up by the cells,
FITC was covalently bonded to AF1, followed by cellular uptake evaluation. The cellular uptake of AF1 readily occurred followed by entry into lysosomes (Fig. S5 in the ESM), similar to the results shown in Fig. 4(c). Thus, AF1 efficiently enhanced the cellular uptake of SeNPs. 3.4
Synergetic anti-cancer effect of AF1-Se
Se is often used for as an anti-cancer treatment [13]. In this study, the anticancer efficacy of AF1-Se was first evaluated against various human cancer and normal cell lines by the MTT assay. Compared with
| www.editorialmanager.com/nare/default.asp
9
Nano Res.
AF1 and SeNP treatment alone (Figs. S6(a) and S6(b) in the ESM), AF1-Se exhibited broad-spectrum inhibition against human breast adenocarcinoma cell lines (MCF-7, MDA-MB-231, and MDA-MB-468) and human hepatocellular carcinoma cell lines (BEL7402, Huh-7, HCCLM9, HepG2, and Hep3B) (Fig. S6(c) in the ESM), and no cytotoxicity against human normal cell lines of HBL-100, L02, and 293T were displayed (Fig. S6(d) in the ESM). The half maximal inhibitory concentration (IC50) is typically used to measure the effectiveness of a substance in inhibiting a specific biological or biochemical function. IC50 is the AF1-Se concentration at which half of the cellular viability is inhibited. The IC50 values of AF1-Se were then estimated for all cell lines as shown in Fig. 5(a). AF1-Se clearly showed the lowest IC50 value of 33 μg·mL−1 against MCF-7 cells, showing some cell selectivity. Notably, selenium concentration was only 14.5 μM in 100 μg·mL−1 AF1-Se through ICP-MS analysis. In other words, the IC50 value of AF1-Se for MCF-7 contains a selenium concentration of only 4.8 μM, which was much lower than the reported value of 7.1 μM [21]. These results
indicated that AF1-Se has a remarkable synergistic anti-cancer effect without toxicity to normal cells. The synergistic effect between AF1 and SeNPs was then analyzed by CompuSyn software using the Chou-Talalay method [43] (Fig. S6 in the ESM). MCF-7 cells were treated with AF1 and SeNPs alone or in combination with a ratio of AF1 and SeNPs of 5.5:1. Quantitative analysis of growth inhibition by isobologram suggested that combination treatment produced an effect greater than individual treatments (Fig. S7(a) in the ESM). As shown in Fig. S7(b) in the ESM, combination indices (CI) characterizing the synergy [44] were 0.015–0.186 at designated concentrations of SeNPs and AF1, indicating strong synergy. These data clearly demonstrate that synergistic interactions between SeNPs and AF1 enhanced the anti-breast cancer effect in vitro. 3.5 AF1-Se induces cell apoptosis and cell cycle arrest in vitro Cancer inhibition of anti-cancer drugs often results from the induction of apoptosis, cell cycle arrest, or a
Figure 5 AF1-Se induces cell apoptosis in cancer cells. (a) IC50 of AF1-Se on selected cancer cells and normal cells (72 h). Cell viability was determined by a colorimetric MTT assay. The data represent the average of at least three independent experiments ± SD. (b) Representative images of DNA fragmentation and nuclear condensation (indicated by arrows) after 24 h-treatment of AF1-Se detected by TUNEL-DAPI co-staining. (c) MCF-7 cells were treated with AF1 (100 μg·mL−1) or AF1-Se (designated concentrations) for 72 h. Cell apoptosis was determined by flow cytometric after annexin V-FITC/PI staining for 15 min at room temperature. The data represent the average of at least three independent experiments ± SD. www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
10
Nano Res.
combination of these processes [45, 46]. Figure 5(b) shows that AF1-Se caused DNA fragmentation (green fluorescence) and nucleus condensation (blue fluorescence) in MCF-7 cells. Flow cytometric analysis was performed to further assess the apoptotic MCF-7 cells induced by AF1-Se. As shown in Fig. 5(c), following treatment with 100 μg·mL−1 of AF1-Se, the number of early apoptotic cells (18.1%) was significantly higher than that of the control group (4.3%). Additionally, the number of late apoptotic MCF-7 cells was much higher than in the control group. Notably, treatment with 100 μg·mL−1 AF1 did not induce apoptosis, which is consistent with our previous results [30]. To gain insight into AF1-Se-induced apoptosis, whole cell extracts were extracted and analyzed by
western blotting. As shown in Fig. 6, activation of caspase-3 as the central regulator of apoptosis [47] and caspase-9 as an initiator of the mitochondriamediated apoptotic pathways [48, 49] were triggered by AF1-Se in a time-dependent manner in MCF-7 cells, indicating that the mitochondria-mediated apoptotic pathway is involved in AF1-Se-induced apoptosis. Poly(ADP-ribose) polymerase (PARP1), a DNA repair enzyme, is downstream of caspase family proteins in apoptosis pathways and serves as a marker of cells undergoing apoptosis [50, 51]. Down-regulation of PARP1 due to cleavage by the caspase family further confirmed the occurrence of earlier mitochondriarelated apoptosis through the caspase-signaling pathway triggered by AF1-Se. Additionally, the expression of
Figure 6 AF1-Se induces activation of intracellular apoptotic signaling pathways and cell cycle arrest in MCF-7 cells. (a) MCF-7 cells were treated with 100 μg·mL−1 of AF1-Se for indicated time intervals and whole cell extracts were prepared, 30 μg of which were resolved on 10% or 12% SDS-PAGE. PARP1, PdCD4, Bcl-2, cleaved-caspase-9, and pro-caspase-3 were detected by western blotting analysis using their specific antibodies with β-actin as a loading control. (b) The digital results were determined by quantitative densitometry. *p < 0.05, **p < 0.001 when compared with the control. (c) MCF-7 cells were treated with 100 μg·mL−1 of AF1-Se for the indicated time intervals, and then incubated with DCFH-DA followed by flow cytometry analysis of the intracellular ROS level. (d) Cell cycle analysis of MCF-7 cells by flow cytometry after treatment with AF1 (100 μg·mL−1) or AF1-Se (designated concentrations) for 36 h, followed by staining with PI. The data represent the average of at least three independent experiments ± SD. | www.editorialmanager.com/nare/default.asp
11
Nano Res.
proteins including Bcl-2 [52] and programmed cell death protein 4 (PdCD4) [53], which are related to cell apoptosis, further confirmed the occurrence of apoptosis. ROS has been suggested to be closely related to cell apoptosis activated by anti-cancer drugs. The high level of ROS can cause damage to macromolecules and further induce cell apoptosis [54, 55]. To determine whether ROS are involved in AF1-Se-induced apoptosis, we examined the induction of cellular ROS production in response to AF1-Se exposure by flow cytometry. The results (Fig. 6(c)) indicate that the mean fluorescence intensity of AF1Se-treated cells ranged from 33.5 to 53.5, while the mean fluorescence intensity of the control group was only 24.3, indicating the potential role of ROS in AF1-Se-induced MCF-7 cell apoptosis. These results demonstrate that AF1-Se induced MCF-7 cell apoptosis through caspase-dependent pathways increase intracellular ROS levels as critical mediators. Taken together, AF1-Se induced apoptosis in MCF-7 cells. The PI staining assay using flow cytometry was conducted to identify the modes of cell antiproliferation by AF1-Se. The average percentage of
cells in S phase increased from 29.7 to 34.6, 39.5, and 51.2% after treatment for 36 h with increasing AF1-Se concentrations (Fig. 6(d)), suggesting that AF1-Se treatment arrested the MCF-7 cell cycle in S phase. Additionally, there were no significant changes in the G2/M phase, and the percentage of G0/G1 phase cells was decreased (Fig. S8(b) in the ESM). Thus, the anti-cancer activities of AF1-Se were caused by a combination of apoptosis and S phase cell arrest. 3.6
In vivo antitumor efficacy
In vivo, we treated MCF-7 xenograft nude mice with AF1 and different doses of AF1-Se to examine their in vivo antitumor efficacy. Based on the progress of tumor volume growth, both AF1- and AF1-Se-treated groups exhibited tumor suppressive effects within 15 days. Moreover, the AF1-Se-treated groups suppressed tumor growth more significantly than AF1treated groups after 15 days (Fig. 7(a)) without body weight loss (Fig. 7(b)). The results showed that AF1-Se significantly inhibited the growth of MCF-7 tumors in a dose-dependent manner, as represented by the decrease in tumor volume and tumor weight (Figs. 7(a)
Figure 7 Anti-tumor activities of AF1 and AF1-Se samples in vivo. (a) Tumor volume in AF1- or AF1-Se-treated xenograft MCF-7 cancer nude mice determined every other day for 21 days. (b) Mean values of mouse body weights. (c) Tumor weights were presented in a scatter plot. (d) Representative photographs of MCF-7 tumors. Bars represent ± SD. *p < 0.05, **p < 0.001 compared to the control. www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
12
Nano Res.
and 7(c)). The inhibition rates of the tumor were approximately 40.6%, 58.2%, and 67.2% for the 5 mg·kg−1 AF1 group, 5 mg·kg−1 AF1-Se group, and 10 mg·kg−1 AF1-Se group, respectively. Moreover, photos of tumors excised from mice in each group (Fig. 7(d)) showed that tumor sizes of all AF1-treated groups and AF1Se-treated groups were visibly smaller than that in the control group. Additionally, no distinct reduction was observed in representative sections of the main organs including the heart, liver, spleen, lung, and kidney, indicating no significant signals of organ damage, inflammatory response, degeneration, and necrosis (Fig. S9 in the ESM). These results demonstrate the effectiveness of the in vivo tumor-suppressed capacity of AF1-Se without cytotoxicity to normal tissues. In accordance with the in vitro analysis, TUNEL positivity (green fluorescence) was barely detectable in tumors of control mice. TUNEL signals were, however, observed in tumors of mice treated with AF1 and AF1-Se (Fig. S10(a) in the ESM). Moreover, tumor tissues treated with a combination of SeNPs and AF1 showed stronger TUNEL signals than those AF1-treated mice. This indicates that a combination of SeNPs and AF1 was a much more efficient strategy for tumor therapy. As shown in Figs. S10(b) and S10(c) in the ESM, after treatment with AF1-Se, the cleavage of caspase-3, caspase-9, and PARP1 was also significantly evoked, and AF1-Se effectively reduced Bcl-2 and dramatically up-regulated PdCD4, demonstrating that the intrinsic apoptotic pathway is also activated by AF1-Se in nude mice. Taken together, these findings indicate that AF1-Se had a potential therapeutic effect in vivo by inducing tumor cell apoptosis. Additionally, the biodistribution and half-life of FITC-AF1 and AF1-Se were evaluated in vivo. Consequently, the half-life of FITC-AF1 in blood circulation was estimated to be 7.5 h from the mean serum concentration–time curve of FITC-AF1 after intraperitoneal injection of 10 mg·kg−1 FITC-AF1 as indicated in Fig. S11 in the ESM. According to fluorescence microscopic observation, FITC-AF1 samples were mainly distributed in the main organs including the heart, liver, spleen, lung, and kidney after peritoneal injection in a time-dependent manner
(Fig. S12 in the ESM). In the heart and liver, green fluorescence reached a maximum at 6 h and decreased over time and disappeared after 7 days. In the lung, spleen, and kidney, abundant green fluorescence appeared after 3 days and minimal green fluorescence was observed on day 7.
4 Conclusion A green and facile method for fabricating hollow nanofibers from a water-soluble stiff polysaccharide (AF1), in which selenium nanoparticles were synthesized (AF1-Se) with enhanced anticancer activity and low toxicity, was developed. Spherical SeNPs with a mean particle size of 46 nm were entrapped in the cavities of AF1 hollow nanofibers through the formation of Se–O bonds, leading to highly stable Se nanoparticles. The cavity of AF1 hollow nanofibers effectively stabilized and protected the Se nanoparticles, leading to their high stability and good dispersion. AF1-Se significantly enhanced the cellular uptake of SeNPs and displayed broad-spectrum cytotoxicity towards various human cancer cells, particularly breast cancer MCF-7 cells, but only minimally harmed normal cells. In vivo animal studies also demonstrated that AF1-Se had significant anti-tumor activities without cytotoxicity to normal tissues. The inhibition ratio of AF1-Se against MCF-7 cells increased from 25% to 75% with an increase of 29 M Se content. The use of AF1 to fabricate well-dispersed Se nanoparticles may be a highly efficient pathway for achieving highly dispersed and stable Se nanoparticles to significantly increase safety, biocompatibility, and antitumor activities with potent applications in the nanomedical materials field.
Acknowledgements This work was supported by the Major Program of National Natural Science Foundation of China (No. 21334005), Major International Joint Research Project (No. 21620102004), the National Natural Science Foundation of China (Nos. 21574102 and 21274114), Special National Key Research and Development Program of China (No. 2016YFD0400202), and New Century Excellent Talents Program of Education Ministry (No. NCET-13-0442).
| www.editorialmanager.com/nare/default.asp
13
Nano Res.
Electronic supplementary material: Supplementary material (more SEM images, SAED images, FT-IR pattern, cellular viability, HE images of main organs and TUNEL images of MCF-7 tumor tissues) is available in the online version of this article at https://doi.org/ 10.1007/s12274-017-1590-7.
References [1] Farokhzad, O. C.; Langer, R. Impact of nanotechnology on drug delivery. ACS Nano 2009, 3, 16–20. [2] Liu, T.; Chao, Y.; Gao, M.; Liang, C.; Chen, Q.; Song, G. S.; Cheng, L.; Liu, Z. Ultra-small MoS2 nanodots with rapid body clearance for photothermal cancer therapy. Nano Res. 2016, 9, 3003–3017. [3] Sarparast, M.; Noori, A.; Ilkhani, H.; Bathaie, S. Z.; El-Kady, M. F.; Wang, L. J.; Pham, H.; Marsh, K. L.; Kaner, R. B.; Mousavi, M. F. Cadmium nanoclusters in a protein matrix: Synthesis, characterization, and application in targeted drug delivery and cellular imaging. Nano Res. 2016, 9, 3229–3246. [4] McNeil, S. E. Evaluation of nanomedicines: Stick to the basics. Nat. Rev. Mater. 2016, 1, 16073. [5] Chen, Q.; Feng, L. Z.; Liu, J. J.; Zhu, W. W.; Dong, Z. L.; Wu, Y. F.; Liu, Z. Intelligent albumin-MnO2 nanoparticles as pH-/H2O2-responsive dissociable nanocarriers to modulate tumor hypoxia for effective combination therapy. Adv. Mater. 2016, 28, 7129–7136. [6] Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer statistics, 2016. CA: Cancer J. Clin. 2016, 66, 7–30. [7] Cruz, L. J.; Que, I.; Aswendt, M.; Chan, A.; Hoehn, M.; Löwik, C. Targeted nanoparticles for the non-invasive detection of traumatic brain injury by optical imaging and fluorine magnetic resonance imaging. Nano Res. 2016, 9, 1276–1289. [8] Barreto, J. A.; O’Malley, W.; Kubeil, M.; Graham, B.; Stephan, H.; Spiccia, L. Nanomaterials: Applications in cancer imaging and therapy. Adv. Mater. 2011, 23, H18–H40. [9] Zhu, X.; Radovic-Moreno, A. F.; Wu, J.; Langer, R.; Shi, J. J. Nanomedicine in the management of microbial infectionoverview and perspectives. Nano Today 2014, 9, 478–498. [10] Mohanraj, V. J.; Chen, Y. Nanoparticles—A review. Trop. J. Pharm. Res. 2006, 5, 561–573. [11] Bao, C. C.; Conde, J.; Pan, F.; Li, C.; Zhang, C. L.; Tian, F. R.; Liang, S. J.; de la Fuente, J. M.; Cui, D. X. Gold nanoprisms as a hybrid in vivo cancer theranostic platform for in situ photoacoustic imaging, angiography, and localized hyperthermia. Nano Res. 2016, 9, 1043–1056.
[12] Venkataraman, S.; Hedrick, J. L.; Ong, Z. Y.; Yang, C.; Ee, P. L. R.; Hammond, P. T.; Yang, Y. Y. The effects of polymeric nanostructure shape on drug delivery. Adv. Drug Deliver. Rev. 2011, 63, 1228–1246. [13] Popova, N. V. Perinatal selenium exposure decreases spontaneous liver tumorogenesis in CBA Mice. Cancer Lett. 2002, 179, 39–42. [14] Wang, H. L.; Zhang, J. S.; Yu, H. Q. Elemental selenium at nano size possesses lower toxicity without compromising the fundamental effect on selenoenzymes: Comparison with selenomethionine in mice. Free Radical Bio. Med. 2007, 42, 1524–1533. [15] Zhang, J. S.; Wang, X. F.; Xu, T. W. Elemental selenium at nano size (nano-Se) as a potential chemopreventive agent with reduced risk of selenium toxicity: Comparison with se-methylselenocysteine in mice. Toxicol. Sci. 2008, 101, 22–31. [16] Liu, W.; Li, X. L.; Wong, Y.-S.; Zheng, W. J.; Zhang, Y. B.; Cao, W. Q.; Chen, T. F. Selenium nanoparticles as a carrier of 5-fluorouracil to achieve anticancer synergism. ACS Nano 2012, 6, 6578–6591. [17] Vekariya, K. K.; Kaur, J.; Tikoo, K. ERα signaling imparts chemotherapeutic selectivity to selenium nanoparticles in breast cancer. Nanomedicine 2012, 8, 1125–1132. [18] Kong, L.; Yuan, Q.; Zhu, H. R.; Li, Y.; Guo, Q. Y.; Wang, Q.; Bi, X. L.; Gao, X. Y. The suppression of prostate LNCaP cancer cells growth by selenium nanoparticles through Akt/Mdm2/AR controlled apoptosis. Biomaterials 2011, 32, 6515–6522. [19] Hu, H. B.; Li, G. X.; Wang, L.; Watts, J.; Combs, G. F., Jr.; Lü, J. X. Methylseleninic acid enhances taxane drug efficacy against human prostate cancer and down-regulates antiapoptotic proteins Bcl-XL and survivin. Clin. Cancer Res. 2008, 14, 1150–1158. [20] Yu, B.; Li, X. L.; Zheng, W. J.; Feng, Y. X.; Wong, Y.-S.; Chen, T. F. pH-responsive cancer-targeted selenium nanoparticles: A transformable drug carrier with enhanced theranostic effects. J. Mater. Chem. B 2014, 2, 5409–5418. [21] Huang, Y. Y.; He, L. Z.; Liu, W.; Fan, C. D.; Zheng, W. J.; Wong, Y.-S.; Chen, T. F. Selective cellular uptake and induction of apoptosis of cancer-targeted selenium nanoparticles. Biomaterials 2013, 34, 7106–7116. [22] Swierczewska, M.; Han, H. S.; Kim, K.; Park, J. H.; Lee, S. Polysaccharide-based nanoparticles for theranostic nanomedicine. Adv. Drug Deliver. Rev. 2016, 99, 70–84. [23] Schepetkin, I. A.; Quinn, M. T. Botanical polysaccharides: Macrophage immunomodulation and therapeutic potential. Int. Immunopharmacol. 2006, 6, 317–333.
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
14
Nano Res.
[24] Wasser, S. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl. Microbiol. Biol. 2002, 60, 258–274. [25] Mizushima, Y.; Yuhki, N.; Hosokawa, M.; Kobayashi, H. Diminution of cyclophosphamide-induced suppression of antitumor immunity by an immunomodulator PS-K and combined therapeutic effects of PS-K and cyclophosphamide on transplanted tumor in rats. Cancer Res. 1982, 42, 5176–5180. [26] Okamura, K.; Suzuki, M.; Yajima, A.; Chihara, T.; Fujiwara, A.; Fukuda, T.; Goto, S.; Ichinohe, K.; Jimi, S.; Kasamatsu, T. et al. Clinical evaluation of schizophyllan combined with irradiation in patients with cervical cancer: A randomized controlled study. Cancer 1986, 58, 865–872. [27] Wu, H. L.; Li, X. L.; Liu, W.; Chen, T. F.; Li, Y. H.; Zheng, W. J.; Man, C. W.-Y.; Wong, M.-K.; Wong, K.-H. Surface decoration of selenium nanoparticles by mushroom polysaccharides-protein complexes to achieve enhanced cellular uptake and antiproliferative activity. J. Mater. Chem. 2012, 22, 9602–9610. [28] Nie, T. Q.; Wu, H. J.; Wong, K.-H.; Chen, T. F. Facile synthesis of highly uniform selenium nanoparticles using glucose as the reductant and surface decorator to induce cancer cell apoptosis. J. Mater. Chem. B 2016, 4, 2351–2358. [29] Xu, S. Q.; Xu, X. J.; Zhang, L. Branching structure and chain conformation of water-soluble glucan extracted from Auricularia auricula-judae. J. Agric. Food Chem. 2012, 60, 3498–3506. [30] Ping, Z. H.; Xu, H.; Liu, T.; Huang, J. C.; Meng, Y.; Xu, X. J.; Li, W. H.; Zhang, L. Anti-hepatoma activity of the stiff branched β-D-glucan and effects of molecular weight. J. Mater. Chem. B 2016, 4, 4565–4573. [31] Xu, S. Q.; Lin, Y.; Huang, J.; Li, Z.; Xu, X. J.; Zhang, L. Construction of high strength hollow fibers by self-assembly of a stiff polysaccharide with short branches in water. J. Mater. Chem. A 2013, 1, 4198–4206. [32] Li, Y. H.; Li, X. L.; Wong, Y.-S.; Chen, T. F.; Zhang, H. B.; Liu, C. R.; Zheng, W. J. The reversal of cisplatin-induced nephrotoxicity by selenium nanoparticles functionalized with 11-mercapto-1-undecanol by inhibition of ROS-mediated apoptosis. Biomaterials 2011, 32, 9068–9076. [33] Osborne, C. K.; Coronado, E. B.; Robinson, J. P. Human breast cancer in the athymic nude mouse: Cystostatic effects of long-term antiestrogen therapy. Eur. J. Cancer Clin. Oncol. 1987, 23, 1189–1196. [34] Xu, H.; Zou, S. W.; Xu, X. J.; Zhang, L. Anti-tumor effect of β-glucan from Lentinus edodes and the underlying mechanism. Sci. Rep. 2016, 6, 28802.
[35] Yang, S. Y.; Wang, C. F.; Chen, S. Interface-directed assembly of one-dimensional ordered architecture from quantum dots guest and polymer host. J. Am. Chem. Soc. 2011, 133, 8412–8415. [36] An, C. H.; Tang, K. B.; Liu, X. M.; Qian, Y. T. Large-scale synthesis of high quality trigonal selenium nanowires. Eur. J. Inorg. Chem. 2003, 17, 3250–3255. [37] Kaur, G.; Iqbal, M.; Bakshi, M. S. Biomineralization of fine selenium crystalline rods and amorphous spheres. J. Phys. Chem. C 2009, 113, 13670–13676. [38] Li, Q.; Chen, T. F.; Yang, F.; Liu, J.; Zheng, W. J. Facile and controllable one-step fabrication of selenium nanoparticles assisted by L-cysteine. Mater. Lett. 2010, 64, 614–617. [39] Duan, H. W.; Kuang, M.; Wang, J.; Chen, D. Y.; Jiang, M. Self-assembly of rigid and coil polymers into hollow spheres in their common solvent. J. Phys. Chem. B 2004, 108, 550–555. [40] Zhou, K. J.; Li, J. F.; Lu, Y. J.; Zhang, G. Z.; Xie, Z. W.; Wu, C. Re-examination of dynamics of polyeletrolytes in salt-free dilute solutions by designing and using a novel neutral-charged-neutral reversible polymer. Macromolecules 2009, 42, 7146–7154. [41] Ma, N. N.; Ma, C.; Li, C. Y.; Wang, T.; Tang, Y. J.; Wang, H. Y.; Mou, X. B.; Chen, Z.; He, N. Y. Influence of nanoparticle shape, size, and surface functionalization on cellular uptake. J. Nanosci. Nanotechnol. 2013, 13, 6485–6498. [42] Wang, F.; Wang, Y.-C.; Dou, S.; Xiong, M.-H.; Sun, T.-M.; Wang, J. Doxorubicin-tethered responsive gold nanoparticles facilitate intracellular drug delivery for overcoming multidrug resistance in cancer cells. ACS Nano 2011, 5, 3679–3692. [43] Chou, T. C.; Talalay, P. Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 1984, 22, 27–55. [44] Chou, T. C. The median-effect principle and the combination index for quantification of synergism and antagonism. In Synergism and antagonism in chemotherapy. Chou, T. C.; Rideout, D. C., Eds.; Academic Press: New York, 1991; pp 61–89. [45] Zheng, S. Y.; Li, X. L.; Zhang, Y. B.; Xie, Q.; Wong, Y.-S.; Zheng, W. J.; Chen, T. F. PEG-nanolized ultrasmall selenium nanoparticles overcome drug resistance in hepatocellular carcinoma HepG2 cells through induction of mitochondria dysfunction. Int. J. Nanomed. 2012, 7, 3939–3949. [46] Wu, S.-B.; Pang, F.; Wen, Y.; Zhang, H.-F.; Zhao, Z.; Hu, J.-F. Antiproliferative and apoptotic activities of linear furocoumarins from Notopterygium incisum on cancer cell lines. Planta Med. 2010, 76, 82–85. [47] Cohen, G. M. Caspases: The executioners of apoptosis. Biochem. J. 1997, 326, 1–16.
| www.editorialmanager.com/nare/default.asp
15
Nano Res.
[48] Tajon, C. A.; Seo, D.; Asmussen, J.; Shah, N.; Jun, Y.-W.; Craik, C. S. Sensitive and selective plasmon ruler nanosensors for monitoring the apoptotic drug response in Leukemia. ACS Nano 2014, 8, 9199–9208. [49] Chen, T. F.; Wong, Y. S. Selenocystine induces caspaseindependent apoptosis in MCF-7 human breast carcinoma cells with involvement of p53 phosphorylation and reactive oxygen species generation. Int. J. Biochem. Cell Biol. 2009, 41, 666–676. [50] Jagtap, P.; Szabó, C. Poly(ADP-ribose) polymerase and the therapeutic effects of its inhibitors. Nat. Rev. Drug Discov. 2005, 4, 421–440. [51] Andrabi, S. A.; Dawson, T. M.; Dawson, V. L. Mitochondrial and nuclear cross talk in cell death. Ann. N. Y. Acad. Sci. 2008, 1147, 233–241.
[52] Oltval, Z. N.; Milliman, C. L.; Korsmeyer, S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell 1993, 74, 609–619. [53] Lu, P.; Sun, H. F.; Zhang, L. X.; Hou, H. L.; Zhang, L.; Zhao, F. Y.; Ge, C.; Yao, M.; Wang, T. P.; Li, J. J. Isocorydine targets the drug-resistant cellular side population through PDCD4-related apoptosis in hepatocellular carcinoma. Mol. Med. 2012, 18, 1136–1146. [54] Chen, T.; Wong, Y. S. Selenocystine induces apoptosis of A375 human melanoma cells by activating ROS-mediated mitochondrial pathway and p53 phosphorylation. Cell. Mol. Life Sci. 2008, 65, 2763–2775. [55] Cairns, R. A.; Harris, I. S.; Mak, T. W. Regulation of cancer cell metabolism. Nat. Rev. Cancer 2011, 11, 85–95.
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research