Photosensitive properties,synergistic antibacterial abilities of intelligent response-type self-assembled nanoparticle TiO 2 @V 2 O 5

Synthesis and characterization of TiO₂@V₂O₅ nanoparticles

According to the previously reported, ³² TiO₂ nanoparticles were prepared by dropwise addition of 3.0 mL of 10% titanium isopropoxide (Aldrich, 99.999%) in anhydrous ethanol to 40 mL of ultrapure water with vigorous stirring. Prior to addition of titanium isopropoxide the pH of the water was adjusted to 1.5, with HClO₄. The solution was kept stirring in a stoppered glass flask. Freshly prepared colloidal suspension was used in all the experiments. The newly prepared ammonium metavanadate solution was vigorously stirred with 200 μL of acetic acid solution was added by dropwise, then stirring for 30 min to make the solution clear and transparent. In accordance with the ratio of titanium dioxide and vanadium pentoxide is 10: 1, added some polyethylene glycol and continue stirring for 30 min, the mixed solution was added to the polyreactor and heated at 200 °C for 6 h. After cooling to room temperature, the obtained pale yellow complex was washed several times with ethanol. Finally, the product was oven dried at 60 °C for 24 h. The untreated TiO₂ nanoparticles were used as controls. All nanoparticles (TiO₂, TiO₂@V₂O₅) were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Conductivity meter, Melvin particle size analyzer, Digital micro ammeter and Energy Dispersive X-Ray Spectroscopy.

Kirby-Bauer disc agar diffusion test

Antibacterial activity determination was tested using E. coil and S. aureus, ATCC 25923, using a testing protocol similar to Kirby-Bauer disc diffusion test in an agar medium. ³³ Bacteria taken from an overnight Mueller-Hinton Broth culture was freshly grown for 4 h having approximately 10⁶ CFU/mL. With this culture, a bacterial lawn was prepared on plate counter agar. Membrane discs (the modified commercial membrane) of 6 mm size were used to observe susceptibility patterns against the different loading of the active layer.

Study of broad spectrum antibacterial properties

The soil obtained from environment was diluted 1000-fold with sterile saline and take 100 μL the above diluent to smear on the fungal and bacterial culture medium, respectively. Thus, acquired environment microorganisms amplify colonies. The diluted solution was treated with TiO₂@V₂O₅ nanoparticles (10 μg/mL) for 2 h and then 100 μL of the above solution was smeared to fungal and bacterial media, respectively.

Conductivity and Coomassie Brilliant Blue G-250 experiments

The permeability of four groups of nanoparticles to E. coil and S. aureus cells was examined. In short, the two bacteria were cultured to logarithmic growth phase, then centrifuged and discard the supernatant, resuspended the precipitate. The same concentrations of nanoparticles were added to those bacterial suspensions, measured their conductivity at different time points at room temperature. Those not added any nanoparticles were set up as a control group, each experiments were repeated three or four times. ³⁴

The E. coil and S. aureus cells were cultured to logarithmic growth phase, then centrifuged and washed for three times, resuspended in 5 mL 20 mmol/L phosphate buffer solution (pH6.0). Then added nanoparticles to those solution respectively and incubation for 2 h. After completion of the reaction, the Coomassie Brilliant Blue G-250 solution was added dropwise to the liquid according to Bradford's method and the protein content was measured at 6 hour, 12 hours, 18 hours, 24 hours respectively. The sterile water were set up as untreated control group, each experiments were repeated three or four times.

Fluorescent-based cell wall/membrane integrity assay and morphological characterization of bacteria

The E. coli and S. aureus cells in log phase were collected by centrifugation (5000 rpm for 5 min) at 4 °C and washing with PBS for three times. The supernatant was discarded and the remaining bacteria were resuspended in 5 mL PBS, then the bacterial solution were divided into four groups and incubated with 100 μL 10 μg/mL nanoparticles for 1 h. After the totally reaction between nanoparticles and bacteria, added 100 μL of fluorescent dyes which were prepared by mixing 10 mg ethidium bromide (EB) and 10 mg acridine orange (AO) in 10 mL phosphate buffer solution (PBS, 0.01 mol/L, pH = 7.4) and stained for 15 min, respectively. After rinsing with PBS for three times, the samples imaged were observed using a Leica TCS SP5 II confocal microscope. The control assay was performed only with TiO₂ nanoparticles.

At first the E. coli and S. aureus cells in log phase cells were incubated with four group nanoparticles for 2 h at 37 °C and the solution concentration was controlled in 10 μg/mL. Then bacterial cells were collected and washed for three times with PBS, followed by immobilization with 2.5% glutaraldehyde solution for 2 h. The samples were dehydrated with sequential treatment of 50, 70, 85, 90, and 100% ethanol for 10 min, gold sputter-coated, and imaged using a scanning electron microscope (SEM; Quanta 200FEG, FEI). ³⁵

Crystal violet dye test and light degradation test

The biofilm mass was quantified by crystal violet staining assay. ³⁶ In the experiments of biofilm destruction by nanoparticles, treatment with both TiO₂@V₂O₅ nanoparticles and illumination resulted in obvious reduction in remaining biofilms. The treatment with just TiO₂, TiO₂@V₂O₅ nanoparticles or both TiO₂ and illumination also displayed certain effect for biofilm destruction compared to control group. According to the above results, we could conclude that TiO₂@V₂O₅ nanoparticles could serve as an efficient agent to produce ROS for the destruction and inhibiting formation of biofilm.

The prepared nanoparticles powder was added to 50 mL methylene blue solution of a certain concentration, and the reaction solution was stirred and irradiated for a period of time. The supernatant was centrifuged and placed in a 721 spectrophotometer to measure the absorbance A_t. (Methyl orange: 465 nm; methylene blue: 664 nm), the degradation rate = (A₀–A_t)/A0 × 100% (A₀, absorbance of the original methylene blue solution; A_t, absorbance of methylene blue solution after degradation)

ROS detection

For total reactive oxygen species (ROS), the bacterial (E. coli and S. aureus) cells were incubated with nanoparticles according to the total ROS assay kit. ³⁷ Briefly, the bacterial cells were harvested by centrifugation, washed twice with PBS, and incubated with 10 mM DCF-DA in PBS at 37 °C for 30 min. The cells were then incubated with nanoparticles at 37 °C for24 h. The intracellular ROS level was examined under a fluorescence microscope with the excitation and emission wavelengths at 495 nm and 525 nm, respectively, and the ROS level was examined by confocal laser scanning microscopy (CLSM) images.

Fluorescent tracking test

The procedure of experiment was carried out using 50 mM H₂O₂, 0.625 mM terephthalic acid, and four groups nanoparticles in 0.2 mM HAc-NaAc buffer (pH 4.0) at 40 °C for 8 h, then used fluorospectrophotometer to track the fluorescent. ³⁸

Cytotoxicity and hemolysis of TiO₂@V₂O₅ nanoparticles

HEK-293 cells were supplied by American Type Culture Collection. ³⁹ Cells were cultured in DMEM containing penicillin (100 UmL⁻¹), streptomycin (100 UmL⁻¹) and 10% fetal bovine serum (FBS) in a humidified incubator at 37 °C and 5% CO₂. Cells were harvested by the use of trypsin and were re-suspended in fresh complete medium before plating. ⁴⁰

Human blood samples stabilized by EDTA were obtained from local hospital. Firstly, 1 mL of blood sample was added to 2 mL of 0.9% NaCl solution, and then red blood cells (RBCs) were isolated from serum by centrifugation at 8000 rpm for 10 min. After being washed five times with 5 mL of 0.9% NaCl solution, the purified blood was diluted to 1/10 of its volume with 0.9% NaCl solution. 0.2 mL of diluted RBC suspensionwas then mixed with, (a) 0.8 mL of D. I. water as a positive control and (b) 100 μg/mL TiO₂@V₂O₅ nanoparticles of 0.9% NaCl as a negative control. Then all the mixtures were vortexed and kept at room temperature for 3 h. After centrifugation at 8000 rpm for 5 min, the absorbance of supernatants at 541 nm was determined by a JASCO V-550 UV-vis spectroscopy.

Plant tissue culture: Miniature roses

The optimum medium was according to the recipe required for the growth of explants. The bud proliferation medium is MS + 6-BA (0.5 mg/L) + NAA (0.1 mg/L), the flowing culture medium is 1/2MS + NAA (0.1 mg/L) and plus 0.8% agar, 3% sugar according to practical requirements, the range of pH was adjusted to 5.8 ∼ 6.2. After the microwave heating for the preparation medium, dispensed into glass bottles about 20 mL. Transfer those preparation medium to autoclaves and sterilization for 20 min at 121 °C. Vitamins and other media which are labile under high temperatures required sterile filter through the shape with a diameter of 0.2 μm. In strict accordance with the aseptic technique to carry out tissue culture, and record the plant growth.

The bud proliferation culture: the stem segments with axillary buds was cut into the lengths about 1.5 cm, chose not obviously broken stem segments as explants and seeded into the first generation medium, then transferred to a thermostatic chamber for culture and carried out induced differentiation. The temperature was maintained at about 25 °C, light intensity 1000 ∼ 2000LX, humidity about 60 to 70%. 7 d for the height record a cycle.

The rooting culture and flowering: when buds grow to about 1.5 ∼ 2 cm, broke off the buds from its basic growth, and transferred them to rooting medium for culture. 10 d for a period, recorded its morphology and flowering.

Characterization of TiO₂@V₂O₅ nanoparticles

Transmission electron microscope (TEM) was used to study the morphology of the nanoparticles. TiO₂ nanoparticles (Figure 2(a)(a1 and a2)) exhibited a wafer size of about 100 nm, after doped with V₂O₅, the size of nanoparticles showed spherical and still about 100 nm (Figure 2(a)(b1 and b2)). In addition, the TiO₂@V₂O₅ nanoparticles have good dispersibility and remain unchanged for 7 days at least (Figure 2(a) (c1 and c2)).

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Figure 2.

(a) TEM of TiO₂ (a1–a2) and TiO₂@V₂O₅ (b1–2; c1–2 (7 d)) nanoparticles. (b) FTIR pattern. (c) XRD pattern. (d) EDX of TiO₂@V₂O₅ nanoparticles. (e) Conductivity of nanoparticles. (*P < 0.05, **P < 0.01) (f) Size distribution. (g) Photo-generated current experiment. (h) Schematic diagram of (g).

Fourier Transform in frared spectroscopy and X-ray diffraction were adopted to affirm the phenomenon of V₂O₅ doped into TiO₂. Due to asymmetric stretching vibration of water of the surface of TiO₂ nanoparticles, the characteristic absorption peak of anatase TiO₂ appeared at about 540 cm⁻¹ and the hydroxyl characteristic absorption peak appeared at 1629 cm⁻¹. In addition, the absorption peak near 1600 cm⁻¹ could be attributed to the bending vibration of N -H, and the sample was prepared by using ammonium vanadate as the source of vanadium, which might be the unreacted residue. The absorption band between 400 ∼ 1000 cm⁻¹ was attributed to the vibration of various vanadium oxygen groups, and the absorption peak near 480 ∼ 600 cm⁻¹ could be attributed to the characteristic absorption peak of crystalline V₂O₅. The absorption peak appeared at 500 ∼ 1500 cm⁻¹, which demonstrated that the dehydration of water molecules on the surface of TiO₂ and V₂O₅ in the synthesis process (Figure 2(b)). The characteristic absorption peak around 25.28° and 20.26° could be derived out the existence of TiO₂ and V₂O₅, respectively. However, the TiO₂@V₂O₅ nanoparticles had the above one characteristic peak which could be attributed to the amount of V₂O₅ were too few to change the x ray diffraction absorption (Figure 2(c)).

Elemental analysis of TiO₂ @ V₂O₅ shows that the content of V element was 5.095%, and the actual proportion of V₂O₅ in TiO₂ @ V₂O₅ was 5.095% × (182/102) = 9.09%. The feeding ratio of TiO₂: V2O₅ was 10: 1, the theoretical ratio of V₂O₅ in TiO₂@V₂O₅ was 9.09%, which was consistent with the actual experimental data. Therefore, it can be proved that V₂O₅ was doped into TiO2 (Figure 2(d)).

TiO₂ is a kind of photosensitive material, which has a certain electrical conductivity under the light, its conductivity can be improve by doping with other materials to some extent. The conductivity of TiO₂@V₂O₅ nanoparticles had greatly improved and the experimental group were better than the untreated group. The above appearance illustrated that the light sensitivity of TiO₂@V₂O₅ nanoparticles could be enhanced by doping with V₂O₅ (Figure 2(e)).

The particle size distribution of the TiO₂@V₂O₅ nanoparticles was more concentrated and stabilized after light treatment and maintained for 7 d at least. However, other groups became agglomeration although they could be redissolved by shaking. Therefore, the particle size and size distribution of TiO₂@V₂O₅ nanoparticles could be maintained by illumination and doping with V₂O₅ (Figure 2(f)).

When the photosensitive material was exposed to light, the light energy could be converted into electrical energy. We pressed the nanoparticles into a sheet and checked the photo-induced charge (electric current) under illumination. Due to the addition of V₂O₅, the yield of the photogenerated charge was higher, which proved that the TiO₂@V₂O₅ nanoparticles were an excellent photosensitive material (Figure 2(g)). The schematic diagram of experimental set-up was shown in Figure 2(h).

Antibacterial activity of nanoparticles

The inhibition zone size was directly proportional to the antibacterial activity, and the regular succession of antibacterial activity was TiO₂@V₂O₅ > TiO₂ >  V₂O₅ >  Control group (sterile saline) in both two strains (Figure 3(a) (a1 to a2)). It could be obviously seen that the control group produced many amplified colonies (Figure 3(b) (b1 to b2)) (Figure 3(c) (c1 to c2)), while the experimental group showed only a few colonies after 5 d (Figure 3(b) (b3)) (Figure 3(c) (c3)), indicating that the TiO₂@V₂O₅ nanoparticles had excellent broad-spectrum antibacterial properties. It showed that the four groups of nanoparticles had antibacterial activity for both E. coil and S. aureus cells suspension to some extent. In short, the sequence of antibacterial activity was TiO₂@V₂O₅ + Light > TiO₂+Light > TiO₂ @V₂O₅ > TiO_2. In addition, the TiO₂@V₂O₅+Light group displayed the best antibacterial activity in particular and its antibacterial ability can maintain for 120 min at least, indicating that the illumination could enhance the antibacterial ability (Figure 3(d)).

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Figure 3.

(a) Kirby-baueerdise diffusion. (b) Fungal broad spectrum antibacterial experiment. (c) Bacterial broad spectrum antibacterial experiment. (d) Time- manner of microbial. (e) Concentration- manner of microbial. (*P < 0.05, **P < 0.01) (f) Conductivity experiments. (g) Coomassie brilliant blue G-250 experiments.(*P < 0.05, **P < 0.01).

Since the antibacterial nanoparticles had concentration-dependent, it could be used as an indicator of antibacterial effect. At the same concentration, the antibacterial effect order of four groups in both E. coil and S. aureus was TiO₂@V₂O₅ + Light > TiO₂ + Light > TiO₂@ V₂O₅ > TiO₂, which showed that the TiO₂@V₂O₅+Light group could reach a wonderful antibacterial effect at a relatively lower concentration and be used as excellent properties of antibacterial agent (Figure 3(e)).

When the antibacterial nanoparticles acted on the bacteria, the bacterial cell membrane would be rupture and the contents will outflow. Therefore, the ion and proteins in bacteria could be used as indicators of the bacteria damage. As time went on, the degree of damage to E. coli and S. aureus would become even more severe. TiO₂@V₂O₅ + Light group was the most obvious one, it could be concluded that it had the strongest antibacterial capacity, which was reflected in the conductivity measurement (Figure 3(f)) and coomassie brilliant blue staining (OD 600 nm) (Figure 3(g)).

Fluorescent-based cell wall/membrane integrity assay and morphological characterization of bacteria

In order to further study the antibacterial mechanism of TiO₂@V₂O₅ nanoparticles, DNA was stained with two fluorescent nucleic acid dyes AO and EB. AO labels both live and dead cells, whereas EB can only penetrate the cells with compromised or damaged membranes (Figure 4(a) and (b)). As shown in TiO₂ groups, there were few dead cells in untreated bacteria, and most cells were alive, as indicated by the green fluorescence. In the E. coli treatment group, almost all the bacteria were stained by EB, and the size of the nanoparticles with antibacterial activity showed a direct association with fluorescence intensity. In all the experimental groups, the antibacterial activity of TiO₂ @ V₂O₅ + Light was the strongest and similar results were found in S. aureus and E. coli. These results indicate that high molecular weight EB dyes can penetrate bacterial cells because of their increased permeability, indicating that damaged cell walls and cell membranes are cell death caused by TiO₂@ V₂O₅ + light exposure.

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Figure 4.

Laser scanning confocal microscope of MDR E. coli (a) and MDR S. aureus (b) after being treated with TiO₂, TiO₂@V₂O₅, TiO₂ + Light, TiO₂@V₂O₅ + Light groups for 60 min, respectively. The assay with TiO₂ NPs is used as the control. The bacterial cells that appeared green (AO)/red (EB) when visualized under fluorescence microscopy were considered live/deadwith intact/damaged membranes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

In summary, the membranes of E. coli and S. aureus cells were intact before treatment with four nanoparticles. SEM images of log phase E. coli and S. aureus cells are shown in (Figure 5(a) and (b)). The E. coli and S. aureus were typically rod- and round-shaped, respectively, both with smooth and intact cell walls. After 2 h incubation of E. coli and S. aureus with 10 mg/mL of the four nanoparticles, the numbers of E. coli and S. aureus greatly decreased and cell walls became wrinkled and damaged. Also the shape and size of the cells changed significantly, indicating that the antibacterial activity of TiO₂@V₂O₅ + Light group was the strongest.

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Figure 5.

Morphological changes of MDR E. coli (a–d) and MDR S. aureus (E–H) with TiO₂, TiO₂@V₂O₅, TiO₂+Light, TiO₂@V₂O₅+Light groups for 60 min, respectively. The assay with TiO₂ NPs is used as the control and visualized with SEM. Arrowheads indicate compromised cell walls.

Mechanism of antibacterial activity

Since the generated ROS exhibited excellent activity toward oxidize nucleic acids, proteins, and polysaccharides in the matrix of biofilm. Mwanwhile, Figure 6(a) showed that TiO₂@V₂O₅ nanoparticles could effectively broke down the existing biofilm and prevent formation of new biofilm (Figure 6(a)).

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Figure 6.

(a) The effect of the TiO₂@V₂O₅ nanoparticles on the biofilm destruction of S. aureus. (a) Pictures of crystal-violet-stained the remaining biofilms. (b) The remaining biofilms were quantified by crystal violet staining. (b) Methyl orange degradation experiments. (c) Methylene blue degradation experiments.

In the absence of illumination, the degradation rate of methyl orange/methylene blue increased slowly with time, and the degradation rate of TiO₂@V₂O₅ nanoparticles were faster than that of TiO₂. However, compared with the irradiation experiment groups, TiO₂@V₂O₅ and TiO₂ (especially TiO₂@V₂O₅) were able to obtain energy from natural light and accelerated the degradation of methyl orange/methylene blue under illumination (Figure 6(b) and (c)). Therefore, TiO₂@V₂O₅ nanoparticles had excellent photocatalytic degradation ability.

Since TiO2 can induce the generation of ROS, in subsequent experiments, we envisioned and determined that the death of the bacteria might be due to oxidative damage.^41,42 By measuring the total ROS concentrations with 20, 70-dichlorofluorescein diacetate (DCFH-DA) and hydroxyl radical (a type of ROS) concentrations with hydroxyphenyl fluorescein (HPF), the results showed that TiO₂@V₂O₅+Light group significantly increase ROS production in of E. coli and S. aureus (Figure 7(a) and (b)). It was likely that the antibacterial activity of TiO₂@V₂O₅+Light group links to the generation of ROS in bacteria. However, TiO₂ group, TiO₂@V₂O₅ group and TiO₂ +Light group did not significantly increase ROS production in E. coli but in S. aureus. These results indicated that one of the mechanisms of the TiO₂@V₂O₅ + Light group to kill bacteria was the production of ROS in bacteria.

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Figure 7.

Cellular total ROS probed with 2,7-dichlorofluorescein diacetate (DCFH-DA) detected with CLSM (a) and PCR (b) (E. coli and S. aureus). The group with TiO₂ NPs wasthe control. (c) Reaction between hydroxyl radical (•OH) and terephthalic acid (TA). (d) Histograms of ΔFL intensity showed the catalysis effect of fourgroup nanoparticles; error bars were taken from three parallel experiments.

The possible mechanism of TiO₂@V₂O₅ nanoparticles peroxidase-like activity may be due to their catalytic ability toward the decomposition of H₂O₂ to generate hydroxyl radical (•OH). We use terephthalic acid (TA) as a fluorescent probe to track •OH to capture •OH and produce 2-hydroxyterephthalic acid (TAOH), which emits a unique fluorescence around 435 nm. After 12 h reaction, compared with control experiments, remarkable fluorescence enhancement at 435 nm indicated the presence of •OH radicals (Figure 7(c) and (d)).

Biocompatibility

The cytotoxicity of the nanoparticles to mammalian cells was also investigated (Figure 8(a)). After treatment with these nanoparticles for 24 h, more than 70% of both HEK 2931 T cell remained viable. Even at very high concentrations (up to 45 μg/mL), the viabilities of both cells remained above 80%, suggesting that these nanoparticles had good biocompatibility and could be used as antibacterial agents in microbial control.

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Figure 8.

(a) Relative viabilities of HEK 293 T cells after being exposed to four types nanoparticles with different concentrations for 24 h. (*P < 0.05, **P < 0.01). (b) UV-vis absorption spectra to detect the presence of hemoglobin in the supernatant of TiO₂@V₂O₅ nanoparticles by using D. I. water and 0.9% NaCl solution as positive and negative controls, respectively. Plant compatibility experiment of Bud proliferation (c) and Flowering (d).

In vitro hemolytic activity was tested in order to assess the interaction between nanoparticles and blood components. Almost no signal around 541 nm in UV-vis absorption spectrum could be detected even at the experimental concentration (Figure 8(b)), which demonstrated that the TiO₂@V₂O₅ nanoparticles were not harmful to blood components.^43,44

Plant tissue culture techniques can be used for the mass production of high quality miniature roses in a short period of time without bacterial or fungal contamination. The three experimental groups showed normal rooting and flowering indicated that those nanoparticles are biosecurity (Figure 8(c) and (d)).