Selenium doped Ni–Ti layered double hydroxide (Ni–Ti LDH) films with selective inhibition effect to cancer cells and bacteria

Abstract

Much attention has been paid to the antibacterial and tumor inhibition effect of the bioelement selenium recently. In this work, different amounts of selenium were doped into the interlayer of Ni–Ti layered double hydroxide (Ni–Ti LDH) films prepared on the surface of nitinol via simple hydrothermal treatment. The biological behaviours of Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, cancer cell line cholangiocarcinoma cells (RBE) and normal cell line human intrahepatic biliary epithelial cells (HIBEpic) cultured on different samples were tested. Results show that doping 3.5% (at%) selenium into the LDH films can effectively inhibit the growth of both kinds of bacteria and cancer cells investigated in this study, but has little adverse effect on normal cells. Such selectivity is verified to stem from the synergistic effect of the doped selenium and the hydroxyl radicals produced by the LDH films.

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1. Introduction

Nitinol is widely fabricated as stents for the palliation treatment of lumen obstruction induced by many kinds of cancers.^1–3 However currently used nitinol stents do not posses bacteria or tumour inhibition ability, and are susceptible to reocclusion by bacteria infection or tumour ingrowth and overgrowth.^4,5 Once reocclusion has occurred, the patients would have to undergo surgery to replace the blocked stent, which will bring huge physical, psychological and economic burdens to the patients. Therefore, developing nitinol stents with selective bacteria and tumour inhibition effect is of great importance.

Layered double hydroxides (LDH), a class of ionic lamellar compound, have been widely applied in the areas of separation,^6,7 catalysis,^8–11 electrochemistry^12–14 and drug carriers.^15–17 Particularly, benefiting from the pH sensitive, biocompatibility and anionic-exchangeable properties, LDHs are especially attractive being employed as biomaterials. Lin et al. synthesised oriented Mg–Fe LDH on the surface of magnesium and found that human mesenchymal stem cells spread better on samples with LDH coated than those on pure magnesium alloy.¹⁸ Yao et al. describe a reliable preparation of Mg–Al LDH micropatterned arrays on gold substrate, which can act both as bioadhesive region for selective cell adhesion and as nanocarrier for drug molecules to regulate cell behaviours.¹⁹ In our previous study, we have successfully fabricated a series of Ni–Ti LDH films with different Ni/Ti ratio on the surface of nitinol which shows selective cancer cell inhibition effect.²⁰ Such selectivity was verified to be determined by the Ni/Ti ratio, films with higher nickel content have stronger inhibition effect to cancer cells. However, nickel is an allergic and cancerous element.^21,22 Considering the stringent safety requirement for biomaterials, the large amounts of nickel release will surely limit the application of Ni–Ti LDH in clinic. Therefore, it is very meaningful to reduce the nickel release amounts of Ni–Ti LDH while do not sacrifice its excellent selective tumour inhibition property. To this end we proposed to dope selenium into the low nickel content Ni–Ti LDH films.

Selenium is an essential trace element in human body. As a component of selenoproteins, selenium has structural and enzymic roles, being best-known as an antioxidant and catalyst for the production of active thyroid hormone.²³ Selenium is essential for the proper function of the immune system, and appears to be a key nutrient in counteracting the development of virulence and inhibiting HIV progress.²⁴ Above all, selenium is considered to have the ability of anticancer^25–27 and antibacterial.^28,29 The mechanism of the inhibition effect of selenium to tumour and bacteria is still being investigated at present. Some studies have shown that the antitumor effect of selenium may be induced by its enhancement to the immune system. Lymphocytes form volunteers supplemented with selenium at 200 μg per day showed an enhanced response to antigen stimulation and an increased ability to develop into cytotoxic lymphocytes which can destroy tumour cells.²⁴ Some other reports showed that the antitumor and antibacterial effect of selenium may be correlated with its monomethylated metabolites, which are likely to show good chempreventive activity.²³

Nowadays, a lot of investigations have been focused on using selenium as an inorganic anticancer and antibacterial agent. Sun et al. synthesized luminescent Ru(II)–thiols protected selenium nanoparticles, which could directly suppress the tumour growth and block blood-vessel growth.²⁵ Wang et al. coated selenium nanoparticles on normal paper towel surfaces through a quick precipitation method, introducing antibacterial properties to the paper towels in a healthy way.²⁹ Chen et al. deposited selenium in TiO₂ nanotubes by electrodeposition, and the resulted TiO₂ nanotubes–Se substrates showed a long term antibacterial and anticancer capacity.³⁰ However, selenium possesses a dual nature, which means too much or too little selenium will both do harm to the health of human beings. Therefore maintain appropriate amount selenium in human body is very important.²⁴ In this study, different amounts of selenium were doped into Ni–Ti LDH, the behaviours of bacteria, cancer cells and normal cells cultured on different samples were investigated. The doping amount of selenium which will kill bacteria and cancer cells while won't affect the normal cells is determined. And the possible anticancer and antibacterial mechanism of the selenium doped Ni–Ti LDH is proposed.

2. Experiment

2.1. Materials preparation and characterization

Synthesis of the nano selenium. 7.896 g selenium powders were put into a reaction vessel with Teflon liner, 100 mL NaOH solution (5 M) was poured into the vessel. The reaction vessel was then sealed and placed in an oven for 12 hours at 80 °C. After the reaction, red sol composed of nano selenium can be got. This process is depicted in Scheme 1(i).

Scheme 1 Schematic diagram of the preparation processes of selenium doped Ni–Ti LDH thin films. (i) The preparation of red selenium sol; (ii) the process of selenium being inserted into the LDH films.

Preparation of the selenium doped Ni–Ti LDH. A commercially available NiTi (50.8 at% Ni) stick was cut into small cylinders with a diameter of 12 mm and thickness of 1 mm. The samples were ultrasonic cleaned in ethanol and deionized water several times and dried in ambient atmosphere for further use. The Se doped Ni–Ti LDH films were prepared by hydrothermal treatment and the process was shown in Scheme 1(ii). Briefly each nitinol plate was immersed in 5 mL NaOH solution (5 M) containing different amounts of selenium sol, and was put in a reaction vessel with Teflon liner at 120 °C for 8 h. The final concentrations of the selenium for different samples were 0 M, 5 × 10⁻⁴ M, 1 × 10⁻³ M and 2 × 10⁻³ M respectively, and the samples were designed as Se0#, Se1#, Se2#, and Se3# accordingly. After the reaction, samples were cooled in room temperature, rinsed with 0.1 M HCl solution and dried in ambient atmosphere.

Characterization of the prepared films. The surface morphologies of the samples were characterized by field emission scanning electron microscopy (FE-SEM; S-4800, HITACHI, Japan) and field emission transmission electron microscopy (FE-TEM; Tecnai G2 F20). Both the films themselves and powders scraped from the films were examined by X-ray diffractometer (XRD; D/Max, Rigaku, Japan) to identify the crystalline phase. The elemental compositions of the films were detected by energy dispersive spectrometer [EDS; equipped on the electron probe X-ray microanalysis system (EPMA, JAX-8100, Japan)]. The valences of the chemical components were determined by X-ray photoelectron spectroscopy (XPS; RBD upgraded PHI-5000C ESCA system, USA) with an Mg Kα (1486.6 eV) source.

Ion release tests. All of the experimental samples (Se0#, Se1#, Se2#, Se3#) were immersed in 10 mL PBS (phosphate buffered saline; pH = 7.4) at 37 °C. After certain periods of time, the extract liquid was collected and the selenium amounts were tested by the inductively-coupled plasma optical emission spectrometry (ICP-OES, Varian Liberty 150, USA).

Electrochemical analysis. CHI760C electrochemical workstation (Shanghai, China) was used to examine the dynamic potential polarization curves and cyclic voltammetry (CV) curves. The electrochemical cell was assembled with a conventional three-electrode system: a graphite rod as the counter electrode, a saturated calomel electrode (SCE) as the reference electrode, and the samples as the work electrode. The polarization curves were tested in a physiological saline solution (0.9% NaCl, pH = 7.0) at room temperature with a scanning rate of 10 mV min⁻¹. The voltammetric measurement was performed in 0.5 M sodium hydroxide solution containing 1 μM glucose at room temperature in a scanning rate of 10 mV min⁻¹. The CV curves of samples in the NaOH solution without glucose were also tested.

Glucose catalysis. Samples were immersed in 1000 ppm glucose solution for five days. The glucose concentration in the resulting solution was determined by the dinitrosalicylic acid kit (DNS). Briefly, 1 mL leaching liquid was taken out and mixed with 2 mL DNS. The resulting solution was boiled in water for 15 min, diluted to 15 mL and examined by an enzyme labelling instrument (BIO-tek, ELX 800) at excitation wavelength of 530 nm. The glucose concentration was calculated according to the standard curve. The DNS was prepared by merging 6.3 g dinitrosalicylic acid, 262 mL sodium hydroxide solution (2 M) and 182 g potassium sodium tartrate into 500 mL hot water. Then the solution was diluted to 1000 mL with ultrapure water and stock in brown bottles for further use.

Hydroxyl radicals detection. Hydroxyl radicals were detected by PTA (pure terephthalic acid) method. Briefly, samples were immersed in 5 mL PTA solution (containing 5 mM PTA and 10 mM NaOH) for 5 days. The photoluminescence (PL) spectra of the resulting solution was then examined by the fluorescence spectrophotometer (HORIBA Jobin Yvon, France, Ex = 310 nm).

2.2. Antibacterial ability evaluation

Bacterial counting method. The antibacterial activity of the specimens was evaluated by bacterial counting method using S. aureus and E. coli. The original bacterium solution was diluted in physiological saline to a final concentration of 10⁷ cfu mL⁻¹. The diluted bacterium solution was then induced to sample surfaces with a density of 60 μL cm⁻², and incubated at 37 °C for 24 h. The dissociated bacterial solution was collected and inoculated into a standard agar culture medium. After culturing for 16 h in the incubator, the pictures of bacterial colonies were taken by a gel imaging system (ProteinSimple, USA). The number of colonies was counted according to the National Standard of China GB/T 4789.2. Bacteria for SEM observation were fixed in 2.5% glutaraldehyde for 4 h at 4 °C. Then the specimens were dehydrated in a series of ethanol solution (30, 50, 75, 90, 95, and 100 v/v%) for 10 min, followed by drying in the hexamethyldisilizane (HMDS) ethanol solution series (33, 50, 66 and 100 v/v%) for 10 min each series sequentially.

Live/dead bacteria staining. Bacteria were cultured on the sample surfaces for 24 h. Afterwards, samples were stained by a LIVE/DEAD BacLight Bacterial Viability Kit (L13152, Molecular Probes) for 15 min in dark, and observed under confocal laser scanning microscopy (CLSM, Leica SP8, Germany).

2.3. Cell viability tests

Cell culture. The cholangiocarcinoma cell line RBE was obtained from Cell Bank of Chinese Academy of Science; human intrahepatic biliary epithelial cells (HIBEpic) were purchased from Sciencell. All of the cell lines were cultured in the media provided by the suppliers, in a humidified atmosphere with 5% CO₂ at 37 °C. Based on the cell condition, cells were passaged at ratio of 1 : 2–1 : 3 every 2–4 days.

Cell proliferation test. Cell proliferation was determined by the alamarBlue assay (AbD Serotec Ltd, UK). Briefly, cells were seeded onto samples with a density of 5 × 10⁴ cell per well. After culturing 1, 4 and 7 days, the culture medium was removed and the samples were rinsed with PBS for 3 times. Then 1.0 mL fresh medium with 10% alamarBlue was added to the samples. After incubation for 4 h, 100 μL of the culture medium was transferred to a 96-well plate and measured by the enzyme labelling instrument at excitation wavelengths of 570 nm and 600 nm. The cell proliferation was calculated according to the instruction of the alamarBlue assay. Cells for SEM imaging were fixed and dehydrated following the same procedures described in Section 2.2.

Live/dead cell staining. Cells were seeded on the specimens with a density of 5 × 10⁴ cell per well and cultured for 4 d. Afterwards, samples were rinsed with PBS and stained by the live–dead staining kit (Biovision, USA) according to the manufacturer's instructions. The stained cells were observed under CLSM.

Nucleus staining. Cells were seeded on the specimens with a density of 1 × 10⁴ cell per well and cultured for 14 d. Then cells were fixed by 4% paraformaldehyde (PFA) diluents for 10 min, followed by PBS rinsing and further stained by DAPI (Chemical International). Images of the stained cells were taken by CLSM. The cell numbers on different samples were obtained by the image processing software Image-Pro-Plus 6.

2.4. Statistical analysis

GraphPad Prism 5 statistical software package was used to analyze data. All of the data were expressed as means ± standard deviation (SD). Statistically significant differences (P) between different groups were got by one-way analysis of variance and Turkey's multiple comparison tests. P < 0.05 was considered to be statically significant, and was represented by the symbol “*”, P < 0.01 was represented by “**”, and P < 0.001 was represented by “***”.

3. Results and discussions

3.1. Preparation of selenium doped Ni–Ti LDH

Fig. 1 show the XRD patterns of the powders scraped from Ni–Ti LDH films doped with various amounts of selenium. A strong reflection peak centred around 11° can be observed from all of the samples. It is the most characteristic peak of LDH which can be assigned to the crystal face of (003). The reflection peaks corresponding to (006), (009), (012), (015), (018), (110), (113) can also be found in the XRD patterns. All of these peaks can be indexed to typical LDH materials.^31–33 Several weak reflections at 2 Theta = 19° and 52° were also observed, which can be assigned to the existence of small amount of nickel hydroxide (PDF #03-0177). The (003) peak of sample Se0# is centred at 12.1°, corresponding to the basal spacing of about 0.73 nm, indicating the interlayer anions of Se0# are hydroxyl ions which have relative small radius. After selenium doping, the (003) peak splits into two overlapping peaks at 11.2° and 12.1°. The appearance of the lower angle peak indicates the interlayer space of LDH become larger (0.78 nm), which may be induced by the insertion of SeO₃²⁻ or SeO₄²⁻ into the LDH layer. With the increase selenium doping amounts, the higher angle peak fades away, illustrating that the interlayer OH⁻ ions are replaced by the doping selenium gradually.

Fig. 1 XRD patterns of sample Se0#, Se1#, Se2# and Se3#.

High resolution TEM images of Se0# is presented in Fig. S1 in the ESI,† both character lattice fringes correspond to Ni–Ti LDH and Ni(OH)₂ can be detected, indicating the coexistence of Ni–Ti LDH and Ni(OH)₂. This result matches well with the XRD patterns.

XPS measurement shows the existence of Ni, Ti, O on all of the treated samples (Fig. S2†). The Ni 2p1/2 and Ni 2p3/2 spin–orbital splitting photo-electrons for Ni–Ti LDH are located at biding energies of 873.7 eV and 856.7 eV. The XPS spectra of Ti 2p indicates two peaks centred at 464.4 eV (Ti 2p1/2) and 458.4 (Ti 2p3/2). The O 1s peak can be divided into two peaks centred at 530.3 eV and 531.5 eV corresponding to oxygen peak in hydroxyl bonding with Ti and Ni respectively. These data are in well agreement with the reported value of Ni–Ti LDH.^20,34 Fig. 2 presents the high resolution selenium XPS spectra for Se1#, Se2# and Se3#. Peaks centred at 59.0 eV and 59.9 eV can be ascribed to the existence of SeO₃²⁻ in the Ni–Ti LDH films, while peak centred at 61.0 eV is corresponding to SeO₄²⁻. Therefore selenium exists mainly as selenate and selenite ions between the LDH layers.

Fig. 2 XPS spectra of Se 3d for sample Se1#, Se2# and Se3#.

The SEM coupled with EDS images of selenium doped Ni–Ti LDH are shown in Fig. S3a.† All of the samples are covered by densely distributed two-dimensional thin nanoflakes with 200 nm in width and length and 20–40 nm in thickness. The surface compositions of the samples examined by EDS are shown in Fig. S3b† and the corresponding data are presented in Table 1. Little content variation of nickel, titanium and oxygen can be detected among different samples, while the selenium content increases along with the rising selenium doping amounts.

Table 1 Surface elemental composition of different samples acquired by EDS

	O (at%)	Ti (at%)	Ni (at%)	Se (at%)
Se0#	22.1 ± 1.10	36.3 ± 0.98	41.5 ± 0.10	0
Se1#	20.5 ± 1.10	34.8 ± 0.54	42.6 ± 0.12	2.1 ± 0.68
Se2#	22.2 ± 1.21	33.1 ± 0.80	41.2 ± 0.47	3.5 ± 0.06
Se3#	24.1 ± 1.91	31.6 ± 0.13	39.7 ± 0.59	4.7 ± 1.19

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The formation process of Ni–Ti LDH had been explained in detail in our previous work.²⁰ Briefly, titanium will release from nitinol in the attack of the high concentration of hydroxyl ions in the solution, resulting in the bond broken between nickel and titanium atom. To lower energy, nickel atoms on nitinol surface will also tend to react with hydroxyl ions in the solution, resulting in the nucleation of nickel hydroxide. The dissolved titanium ions in the solution then transport to the lattice of nickel hydroxide and replace nickel, leading to the generation of Ni–Ti LDH.

In order to only incorporate selenium in the Ni–Ti LDH lattice, nano-selenium was chose as the dopant. Fig. S4† shows the SEM and EDS images of the nano-selenium fabricated in this study. It can be found that the nano-selenium shows spherical morphology with a diameter of about 500 nm. The nano-selenium sol is red in colour and shows Tyndall effect (Fig. S5†) just in accordance with the previous report.^25–27 The nano-selenium was prepared by hydrothermal treatment. During the treatment, the elementary selenium will react with the sodium hydroxide as follows:

3Se + 6NaOH ↔ 2Na₂Se + Na₂SeO₃ + 3H₂O (1)

This reaction will be accelerated at high temperature and pressure under hydrothermal condition, resulting in the thoroughly dissolved of selenium powder in the sodium hydroxide solution. However, this reaction is reversible and elemental selenium can nucleate and growth into nano particles because of the reverse reaction. Large amounts of hydroxyl ions will be absorbed on the surface of the newly formed nano-selenium, resulting in the generation of electrostatic repulsion between different selenium particles. Therefore, the nano-selenium won't coagulate but form stable nano-selenium sol.

Selenium doped Ni–Ti LDH films were prepared by hydrothermally treating nitinol in the nano-selenium containing sodium hydroxide solution. In this condition selenium will turn to Se²⁻ and SeO₃²⁻ as shown in formula (1). Theses anions will then enter into the interlayer gap of Ni–Ti LDH as charge compensation ions. Se²⁻ and SeO₃²⁻ have strong reducing property and will react with oxygen in the atmosphere, leading to the formation of SeO₄²⁻. The reaction formula is shown below:

2Se²⁻ + 3O₂ → 2SeO₃²⁻ + O₂ ↔ 2SeO₄²⁻ (2)

3.2. Physical–chemical property of Se doped Ni–Ti LDH thin films

Fig. 3 shows the selenium release profile and the corresponding release fractional ratio of different samples. A sustained selenium release can be observed from all of the selenium containing samples. And the selenium release amounts increase with the selenium doping amounts. About 30 percents selenium was released in one day for all of the samples. The obvious burst release may be induced by the selenium ions physically adsorbed on the surface of the films. In the preparation process of the Se doped Ni–Ti LDH films, not all of the selenium ions in the environment will enter into the interlayer of LDHs. The interlayer selenium ions will keep going out to the outside environment, while the selenium ions in the environment will continuously enter into the interlayer space, and reach a dynamic balance at last. Therefore, there is always some selenium ions left in the environment. Some of these selenium ions will adsorbed on the surface of the LDH film. Due to the weak binding force between the physically adsorbed selenium ions and the LDH film, these selenium ions will preferentially release in the initial stage, resulting in the burst release. After immersing for 15 d, only about 60 percents selenium was released from different samples, indicating the selenium releasing can maintain for a long time. As selenium play an important role in a lot of biological function. The released selenium is considered to have a huge impact on cell and bacteria behaviours.

Fig. 3 Cumulative selenium release amounts (a) and fraction (b) of different samples in PBS.

Corrosion resistance is another important property for biomaterials. In this study Tafel curves were tested to characterize the corrosion resistance of different samples. Results are presented in Fig. S6.† Se0# has the lowest corrosion potential and the highest corrosion current which means Se0# is the easiest to be corroded. The corrosion resistance of the samples increase along with the increase of selenium doping amounts. This result indicates the interaction between selenium and the LDH layers is stronger than the LDH–OH interaction. Therefore, the replacement of interlayer hydroxyl ions by selenium will result in a more stable structure.

CV curves of different samples tested in the solution with and without glucose are shown in Fig. 4a, the cathodic and anodic peaks assigned to the Ni²⁺/Ni³⁺ redox can be found from all of the prepared samples.^35,36 After adding glucose to the electrolyte, an obvious increase of cathodic peak can be detected, which means all of the samples has the ability to catalyze the oxidation of glucose. The quantitative analysis of the glucose oxidation ability of different samples is shown in Fig. 4b. Among all of the prepared films, Se0# and Se1# has the highest capacity to catalyze the oxidation of glucose, while with the increase of selenium doping amounts, the catalysis ability of the samples recede. According to previous study, the catalysis function of Ni–Ti LDH is based on the reaction below:³⁷

Ni²⁺ + OH⁻ − e⁻ ↔ Ni³⁺ + H₂O ↔ Ni²⁺ + OH˙ (3)

OH˙ + organic compound → product (4)

Fig. 4 Cyclic voltammetry curves of different samples in the existence and without glucose (a). Residual glucose content of the glucose solution with different samples soaking in (b). Photoluminescence spectra of the PTA solution after different samples being soaked in for 5 days (c).

Therefore, the catalysis ability of Ni–Ti LDH is originated from the production of hydroxyl radical. In the existence of selenium, hydroxyl radical will preferentially react with selenium (formula (5)), less hydroxyl radicals will be left, so the catalysis ability of the materials was attenuated.

OH˙ + SeO₃²⁻ → SeO₄²⁻ + H₂O (5)

To verify the hydroxyl radicals produce ability of different samples, PTA method was used. The results are presented in Fig. 4c. Sample Se0# shows the strongest ability to produce hydroxyl radicals. With the increase doping amounts of selenium, less and less hydroxyl radicals can be detected from the samples. These results are in well agreement with the discussion above. The generation and consumption processes of the hydroxyl radicals are depicted in Scheme 2.

Scheme 2 Schematic diagram of the production of hydroxyl radicals by Ni–Ti LDH (i), and their consumption by the doped selenium between the Ni–Ti LDH layers (ii).

3.3. Antibacterial ability

Fig. 5 shows the SEM images of S. aureus and E. coli cultured on different samples. With regard to S. aureus, a large amount of bacteria can be found on the surface of NiTi. While, on the LDH films, the amounts of the bacteria obviously decreased, indicating the LDH films are cytostatic or cytocidal to S. aureus. The situation is different for E. coli. There are still a lot of E. coli can be observed on the sample Se0#, which means the LDH films show no inhibition effect to this kind of bacteria. However, after doping selenium into the films, the samples start to show strong antibacterial ability. E. coli can hardly be detected on the selenium doped samples, besides almost all of the bacterial membranes are greatly damaged. To further evaluate the antibacterial ability of the prepared films, bacteria counting method and live dead staining were applied to S. aureus and E. coli cultured on different samples. Fig. 6 and 7 shows the typical photos of the number of bacteria colonies on different samples and the CLSM images of the live/dead stained bacteria respectively. The colony number of both types of bacteria on selenium containing samples is much lower than that on NiTi. Besides more dead bacteria can be detected on the selenium containing films compare to that on NiTi. However, S. aureus and E. coli react differently to the LDH films without selenium doping. The amounts of S. aureus on sample Se0# are largely reduced but nearly no amounts change of E. coli can be detected compare to that on NiTi. The live/dead staining images show that a lot of dead S. aureus can be found on Se0# but dead E. coli can be hardly detected on the same sample. These results indicating that the selenium doping LDH films are able to inhibit the growth of both types of bacteria but without selenium the LDH films only show inhibition effect to S. aureus.

Fig. 5 SEM images of S. aureus (a-n, b-n, c-n, d-n, e-n) and E. coli (f-n, g-n, h-n, i-n, j-n) cultured on different samples at low (n = 1) and high (n = 2) magnification.

Fig. 6 Photos of re-cultivated S. aureus (a-n) and E. coli (b-n) colonies on agar which have been previous dissociated from different samples [n = 1, 2, 3, 4, 5 represent NiTi, Se0#, Se1#, Se2# and Se3# respectively].

Fig. 7 The CLSM images of live/dead stained S. aureus (a) and E. coli cultured on different samples (b).

The antibacterial ability of the selenium containing LDH films is supposed to result from the combined effects of the hydroxyl radicals and the selenium released from the films. As verified above, the LDH films can produce a large amount of hydroxyl radicals. The hydroxyl radicals may induce oxidative stress and lead to the death of bacteria.^38,39 But different types of bacteria have different sensitivities to hydroxyl radicals. Fig. S7a† shows the effect of hydroxyl radicals to bacteria activity. It can be found that S. aureus is more sensitive to hydroxyl radicals than E. coli. The IC50 value of hydroxyl radicals to S. aureus is about 200 μM, while the value for E. coli is 10 000 μM. The amounts of hydroxyl radicals produced by the LDH films may be enough to kill S. aureus but too few to inhibit the growth of E. coli. Therefore, the sample Se0# can inhibit S. aureus but has no adverse effect to E. coli. To endow the sample with broad-spectrum antibiosis property, the antibacterial element selenium was doped into the LDH films.^40,41 Fig. S7b† shows the IC50 curves of selenium to S. aureus and E. coli. The results indicated that E. coli is very sensitive to selenium with an IC50 value of 300 μM. While the IC50 value of selenium to S. aureus is more than 1000 μM. Therefore, after doping selenium into the films, the E. coli can also be effectively inhibited.

3.4. Anticancer ability

Human cholangiocarcinoma cells (RBE) and human intrahepatic biliary epithelial cells (HIBEpic) were seeded on different samples to investigate the different effects of the selenium doped LDH films to cancer cells and normal cells. The cell proliferation was characterized by alamarBlue assay, and the results are presented in Fig. 8a and b. Se0# has the ability to inhibit the growth of cancer cells. However, after doping 2.1% selenium (at%) into the films (Se1#), the cancer cell inhibition effect of the LDH films disappear. Continue to increase the selenium doping amounts, the films become cytostatic to cancer cells again. While, with regard to normal cells, the selenium doping films present no obvious cytotoxicity until 4.7% selenium (at%) was added into the LDH films (Se3#). The SEM images of cells cultured on different samples for 4 d presented in Fig. 8c. Cancer cells on sample Se0#, Se2# and Se3# show spindle morphology, indicating cells on these samples are in poor state. While cancer cells on NiTi and Se1# are well spread and nearly covering the whole surface of the samples. Situation are different for normal cells, it can be found that all of the samples are covered by normal cells completely except for Se3#. The CLSM images of live/dead stained cells are presented in Fig. 9, more dead cancer cells can be found on the surfaces of Se0#, Se2# and Se3# than that on NiTi and Se1#. While for normal cells, nearly no dead cells can be detected from all of the sample surfaces until 4.7% selenium (at%) was doped into the LDH films. To characterize the long-term effect of the Se doped LDH films to cells, we prolonged the cell cultivation time to 2 weeks. Cells were stained by DAPI and observed under CLSM. The results are shown in Fig. 10. It can be found that samples Se0#, Se2# and Se3# show obvious inhibition effect to cancer cells even after 2 weeks' cultivation, while only Se3# is cytostatic to normal cells. These results indicate that the selenium doping LDH films have different effects to cancer cells and normal cells. By adjusting the selenium doping amounts, selective control of cellular behaviour can be realized. In this study, we found that the sample Se2# can effectively inhibited the growth of cancer cells but have little adverse effect to normal cells. Such property makes Se2# a promising material in the palliation treatment of cancer.

Fig. 8 Reduction percentage of alamarBlue for RBE cells (a), and HIBEpic cells cultured on different samples for various periods of time. The SEM images of RBE cells and HIBEpic cells cultured on different samples for 4 days (c).

Fig. 9 The CLSM images of live/dead stained RBE cells (a) and HIBEpic cells (b) cultured on different samples for 4 days.

Fig. 10 The CLSM images of nucleus stained RBE cells and HIBEpic cells cultured on different samples for 2 weeks (a); and the corresponding quantification results of the number of cancer cells (b) and normal cells (c).

The different effect of the selenium doped LDH films to cancer cells and normal cells may be also stemmed from the combined function of the hydroxyl radicals and the selenium released from the samples. As shown in Fig. S8a,† cancer cells are more sensitive to hydroxyl radicals than normal cells. Therefore, the sample Se0# can inhibit the growth of cancer cells but have no inhibition effect to normal cells. As discussed in Section 3.2, doping selenium into LDH films will decrease the hydroxyl radicals' production, the cytotoxic effect of the LDH films to cells will thereby recede. Therefore, sample Se1# show little adverse effect to both cancer cells and normal cells. However, with the increase selenium doping amounts, more selenium ions will release from the films. Previous study have shown that selenium is an cytotoxic element at high concentration.^42,43 In consequence, the sample Se3#, doped with the highest amounts of selenium, show obvious inhibition effect to both cancer cells and normal cells. With regard to sample Se2#, its selective tumour cell inhibition effect may be resulted from the differences in selenium sensitivity of tumour cells and normal cells. As shown in Fig. S8b,† the cancer cell line RBE is more sensitive to selenium than the normal cell line HIBEpic. Therefore, in the existence of the same amount of selenium, the growth of cancer cells may have been inhibited while the normal cells will not be affected.

4. Conclusions

Selenium doped Ni–Ti LDH films were prepared via simple hydrothermal treatment. These films can release hydroxyl radicals and selenium ions at the same time, and the release amounts can be adjusted by the doping amounts of selenium. With the increase of selenium doping amounts, the amounts of hydroxyl radicals released from the films decrease while selenium released from the films increase. Under the combined function of hydroxyl radicals and selenium ions, the behaviour of different types of bacteria, cancer cells and normal cells can be selectively controlled. In this study we found that LDH films doped with 3.5% selenium (at%) can effectively inhibited the growth of both types of bacteria (S. aureus and E. coli) and cancer cells but have little adverse effect to normal cells. Such specific properties of the prepared films make them show great potential in the palliation treatment of many kinds of cancers.

Acknowledgements

Financial support from the National Basic Research Program of China (973 Program, 2012CB933600), National Science Fund for Distinguished Young Scholars (51525207), National Natural Science Foundation of China (51401234), Shanghai Committee of Science and Technology, China (14XD1403900) are acknowledged.

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Footnotes

† Electronic supplementary information (ESI) available: TEM images of Se0#; XPS spectra, SEM images of surface and cross section, EDS and XRD patterns of the prepared films. Elementary composition of different samples acquired by XPS tests; SEM image, EDS pattern and photo of the prepared nano selenium sol; Tafel curves of different samples; IC50 curves of H₂O₂ and selenium to S. aureus, E. coli, cancer cells and normal cells; contact angels of different samples; CLSM images of the cytoskeleton stained cancer cells and normal cells cultured on different samples. See DOI: 10.1039/c5ra18740k

‡ These authors contributed equally to this work.

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