Bactericidal effects and accelerated wound healing using Tb₄O₇ nanoparticles with intrinsic oxidase-like activity

Abstract

Background

Nanomaterials that exhibit intrinsic enzyme-like characteristics have shown great promise as potential antibacterial agents. However, many of them exhibit inefficient antibacterial activity and biosafety problems that limit their usefulness. The development of new nanomaterials with good biocompatibility and rapid bactericidal effects is therefore highly desirable. Here, we show a new type of terbium oxide nanoparticles (Tb₄O₇ NPs) with intrinsic oxidase-like activity for in vitro and in vivo antibacterial application.

Results

We find that Tb₄O₇ NPs can quickly oxidize a series of organic substrates in the absence of hydrogen peroxide. The oxidase-like capacity of Tb₄O₇ NPs allows these NPs to consume antioxidant biomolecules and generate reactive oxygen species to disable bacteria in vitro. Moreover, the in vivo experiments showed that Tb₄O₇ NPs are efficacious in wound-healing and are protective of normal tissues.

Conclusions

Our results reveal that Tb₄O₇ NPs have intrinsic oxidase-like activity and show effective antibacterial ability both in vitro and in vivo. These findings demonstrate that Tb₄O₇ NPs are effective antibacterial agents and may have a potential application in wound healing.

Electronic supplementary material

The online version of this article (10.1186/s12951-019-0487-x) contains supplementary material, which is available to authorized users.

Background

Wound infection is an important cause of poor wound healing and its treatment often requires the use of antibiotics [1, 2]. However, excessive use of antibiotics may lead to the development of antibiotic-resistant bacteria, and may also cause side effects on human health, such as gastrointestinal disturbances. In recent years, developments in nanomaterial technology have provided an opportunity to develop novel antimicrobial agents. Due to the diversity in mechanisms of action against bacteria, bacterial cells are less likely to develop antibacterial resistance compared to existing antibiotics [3–5]. However, most of these nanomaterials have application limitations, such as cytotoxicity, not biocompatible for human use, and environmental concerns.

Nanozymes are nanomaterials that catalyze the same reactions originally catalyzed by natural enzymes in biological systems [6–8]. Over the past several years, a wide variety of nanomaterials, such as noble metals [9–11], metal oxides [12–14], and carbon nanomaterials [15–18], have been explored as potential nanozymes. Based on their intrinsic enzyme-like activity, several nanozymes have been used in antibacterial applications [19–23]. For instance, platinum nanomaterials have shown effective antibacterial activity in the presence of hydrogen peroxide (H₂O₂) [24]. The antibacterial activity of these nanozymes are attributed primarily to their oxidase- and peroxidase-like activities that catalyze the production of hydroxyl radicals (·OH) in the presence of exogenous H₂O₂ and enhance the cellular levels of reactive oxygen species (ROS) within bacteria cells. Fang et al. also showed that palladium nanomaterials with oxidase- and peroxidase-like activities displayed effective antibacterial activity in the presence of H₂O₂ [25]. Although many reported enzyme-like nanomaterials have been proposed as novel antibacterial agents, the high price and persistence in living tissues are still important issues. Moreover, the application of H₂O₂ in human wound disinfection is harmful to healthy tissue and may delay wound healing [26].

Terbium oxide nanoparticles (Tb₄O₇ NPs) have been extensively used as precursors for the synthesis of lanthanide nanophosphors and superconductor materials [27, 28]. For example, Tb₄O₇ complexed with reduced graphene oxide composite exhibit typical green emission of terbium ions as well as the blue self-luminescence of graphene [28]. In addition, it has been found that Tb₄O₇ NPs can be used as analytical reagents for food analysis [29]. Compared with noble metal nanomaterials, Tb₄O₇ NPs are easier to synthesize and are less expensive. However, a review of scientific literature was unable to find any studies that described the enzyme-like activity of Tb₄O₇ NPs and their applications as antibacterial agents. In this paper, we show that Tb₄O₇ NPs have an intrinsic oxidase-like activity at acidic pH values, as they quickly oxidize a series of organic substrates in the absence of H₂O₂. We then demonstrate the relationship between the oxidase-like property of Tb₄O₇ NPs and their antibacterial activity with in vitro studies. Finally, the effects of Tb₄O₇ NPs on wound disinfection and healing are evaluated in in vivo studies using a wound infection mouse model.

Materials and methods

Chemicals and materials

Tb₄O₇ NPs were purchased from US Research Nanomaterials, Inc. (TX, USA). 3,3,5,5-tetramethylbenzidinedihydrochloride (TMB), diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), o-phenylenediamine (OPD), and Lipid Peroxidation MDA Assay Kit were all purchased from Sigma-Aldrich (St. Louis, MO). The Live/Dead BacLight bacterial viability kit and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) were obtained from Thermo Fisher Scientific, Inc. (MA, USA). 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) and Cell Counting Kit-8 (CCK-8) were obtained from Dojindo Laboratories (Kumamoto, Japan). Escherichia coli (E. coli) and Staphylococcus aureus subsp. aureus (S. aureus) were obtained from the China General Microbiological Culture Collection Center (CGMCC, Beijing, China). Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, MD, USA).

Characterization of Tb₄O₇ NPs

The hydrodynamic size and zeta potential of the Tb₄O₇ NPs were measured using a Zetasizer Nano-ZS (Malvern, UK). The morphology and size of the Tb₄O₇ NPs were characterized using a transmission electron microscopy (TEM, Tecnai G-20, FEI). The UV–vis absorption spectrum was recorded on a spectrophotometer (UV-3600, Shimadzu).

Electron spin resonance spectroscopic measurements

The electron spin resonance (ESR) measurements were carried out using a Bruker EMX ESR spectrometer according to our previous study [3, 9]. The final concentration of each component is described in each figure caption. All the ESR measurements were carried out at ambient temperature.

Measurement of intracellular ROS

After Tb₄O₇ NPs (100 μg/mL) treatment, bacteria (1 × 10⁹ CFU/mL) were collected by centrifugation and incubated with DCFH-DA (10 µM) for 30 min at dark, and stained bacteria were visualized with a confocal laser microscopy.

Cytotoxicity experiments

HUVECs were employed for investigating the cytotoxicity of Tb₄O₇ NPs. HUVECs were seeded at a density of 1 × 10⁵ cells/well in 96-well plates and incubated overnight. The HUVECs were then incubated with Tb₄O₇ NPs (0–100 μg/mL) for 24 h, and cell viability was measured by MTT assay.

Mice injury model

BALB/c mice (8 weeks) were purchased from Pengsheng. On the day 0, the mice were anesthetized using 10% chloral hydrate. Then, the dorsal hair of mouse was shaved, full-thickness skin wounds with the diameter of 10 mm were created on the back of each mouse. After 24 h (day 1), the mice were treated with 50 μL PBS or Tb₄O₇ NPs (100 μg/mL). The Tb₄O₇ NPs were dripping on the surface of the wound.

Hemolysis test

Fresh blood was collected under sterile conditions from healthy BALB/c mice (n = 5) into an anticoagulation tube. The red blood cells were precipitated by centrifugation at 2000 rpm for 10 min and washed three times with PBS buffer solution to obtain red blood cells. The appropriate amount of red blood cells was diluted five times with PBS buffer solution to prepare a red blood cell solution. 20 μL of the diluted red blood cell suspension was mixed with a series of different concentrations of Tb₄O₇ NPs (0–200 μg/mL); ultrapure water was used as control. All the above samples were incubated at 37 °C for 2 h, centrifuged at 2000 rpm for 10 min, imaged, and the supernatant after centrifugation was taken in a 96-well plate to measure the absorbance at 540 nm using a microplate reader. The hemolysis rate was calculated as follows:

$$\text{Hemolysis rate}\left(\%\right)=\left(\text{sample absorption}-\text{negative control absorption}\right)/\left(\text{positive control absorption}-\text{negative control absorption}\right)\times 100\%,\text{and hemolysis rate exceeding 5}\%\text{is considered hemolysis}.$$

Results and discussion

Characterization of Tb₄O₇ NPs

The Tb₄O₇ NPs used in the present study were purchased from US Research Nanomaterials, Inc. The physical characterization of Tb₄O₇ NPs is shown in Additional file 1: Figure S1 and included images of particle core size and shape captured by TEM, the mean and homogeneity of particle hydrodynamic size by dynamic light scattering (DLS), and particle absorption spectrum by UV–vis. According to TEM and DLS data, the dispersity of Tb₄O₇ NPs is poor. The mean core particle size of Tb₄O₇ NPs is approximately 200 nm (Additional file 1: Figure S1a, b); while the DLS result of Tb₄O₇ NPs is around 400 nm (Additional file 1: Figure S1c). This is mainly due to the size measured by DLS was a hydrodynamic size, and therefore the nanoparticles showed a larger hydrodynamic volume due to solvent effect in the hydrated state. The zeta potential value of Tb₄O₇ NPs is 31.6 mV in water. The UV–vis spectrum of Tb₄O₇ NPs is shown in Additional file 1: Figure S1d.

Catalytic activity of Tb₄O₇ NPs as oxidase mimetics

The oxidase-like activity of Tb₄O₇ NPs was evaluated using the substrate TMB. The UV–vis spectroscopy measurements show time-dependent increases in TMB oxidation catalyzed by Tb₄O₇ NPs, yielding a blue-colored product (Fig. 1a, b). In addition, Tb₄O₇ NPs can also catalyze the oxidation of ABTS and OPD (Fig. 1a).

Fig. 1.

Oxidase-like activity of Tb₄O₇ NPs. a A photograph showing the capability of the Tb₄O₇ NPs in catalyzing the oxidations of TMB, ABTS, and OPD that produce colored products. b Time-dependent absorption spectra of TMB catalyzed by Tb₄O₇ NPs. c Absorbance at 652 nm measured from samples containing TMB and different concentrations of Tb₄O₇ NPs. d The specific activities of Tb₄O₇ NPs. Steady-state kinetic assays of Tb₄O₇ NPs (e, f). e TMB concentration dependence of initial reaction velocity. f Double-reciprocal plot generated from (d)

We used the oxidation of TMB as a model reaction and found that the catalytic efficiency of the Tb₄O₇ NPs is dependent on TMB concentrations, pH and temperature (Fig. 1c and Additional file 1: Figure S2). As shown in Additional file 1: Figure S2a, Tb₄O₇ NPs exhibit excellent catalytic activity over a broad temperature range (25–60 °C). Moreover, an acidic condition (pH = 3.6) is conducive to the oxidase-like activity of Tb₄O₇ NPs (Additional file 1: Figure S2b). We adopted pH 3.6 and 25 °C (room temperature) as the standard conditions for subsequent studies.

Next, we determined the apparent steady-state kinetic parameters for the reaction of Tb₄O₇ NPs with TMB. Typical Michaelis–Menten curves were established (Fig. 1d). The curves were then fitted to the double-reciprocal Lineweaver–Burk plots (Fig. 1e), from which the kinetic parameters shown in Table 1 were determined.

Table 1.

The kinetic constants of Tb₄O₇ NPs

	[E] (M)	Km (M)	Vmax (M/s)	Kcat (s−1)	Kcat/Km (M−1 S−1)
Tb4O7 NPs	7.04 × 10−10	1.24 × 10−4	4.31 × 10−8	1.61 × 10−4	1.30

[E] is the concentration of Tb₄O₇ NPs. The particle number of Tb₄O₇ NPs is calculated using the density and diameter of Tb₄O₇ NPs. Dividing the particle number by the Avogadro constant is the molar concentration of Tb₄O₇ NPs

Effects of Tb₄O₇ NPs on the anti-oxidant defense system

The above results show that Tb₄O₇ NPs have oxidase-like activity oxidizing TMB, ABTS, and OPD in the absence of H₂O₂. We predict that Tb₄O₇ NPs would deplete intracellular antioxidants and, eventually, disrupt the antioxidant defense systems of bacteria. To test this hypothesis, we examined the effects of Tb₄O₇ NPs on ascorbic acid (AA) oxidation in vitro. AA is an important endogenous bacterial antioxidant that prevents cellular damage from ROS. AA can be oxidized to form an intermediate ascorbyl radical (·AA), which is detectable by ESR spectroscopy [9]. As shown in Fig. 2a, the oxidation of AA was negligible within 10 min, while in the presence of Tb₄O₇ NPs the system showed a time-dependent increase in the ESR signal intensity in the first 8 min and then decreased over time. These results indicate that Tb₄O₇ NPs can accelerate AA oxidation.

Fig. 2.

a Oxidation of AA by Tb₄O₇ NPs. b ESR spectra of BMPO/·OH generated from a sample solution containing 25 mM BMPO, 1 mM H₂O₂ in the absence (control) and presence of different concentrations of Tb₄O₇ NPs

In the substrate oxidation mechanism of most oxidases in nature, oxygen acts as the electron acceptor and is reduced to H₂O₂. To gain a better understanding of the oxidation of AA by Tb₄O₇ NPs, we examined whether the catalytic oxidation product of Tb₄O₇ NPs and AA produced H₂O₂. As shown in Additional file 1: Figure S3, a marked increase of H₂O₂ was detected in the presence of Tb₄O₇ compared to control (AA alone). Moreover, the production of H₂O₂ was Tb₄O₇ concentration-dependent.

Previous studies have demonstrated that several kinds of nanoparticles are capable of catalyzing the production of hydroxyl radicals by H₂O₂ [30, 31]. Therefore, we determined whether Tb₄O₇ NPs would catalyze the production of hydroxyl radicals by H₂O₂ using ESR spectroscopy. We selected BMPO as the capture agent, since BMPO can capture hydroxyl radicals to form BMPO/·OH adducts indicated by the presence of four characteristic lines on the ESR spectrum. As shown in Fig. 2b, the characteristic ESR signals of BMPO/·OH were negligible in the absence of Tb₄O₇ NPs. However, the addition of Tb₄O₇ NPs resulted in a strong ESR spectrum that displayed the four characteristic lines (1:2:2:1) of BMPO/·OH. These results clearly show that Tb₄O₇ NPs can be used as a catalyst in the decomposition of H₂O₂ to produce hydroxyl radicals.

Taken together, our results confirm that Tb₄O₇ NPs are capable of catalyzing the oxidation of biologically relevant antioxidant agents, resulting in the production of H₂O₂. Moreover, Tb₄O₇ NPs can further catalyze the production of hydroxyl radicals via the decomposition of H₂O₂.

Antibacterial activity of Tb₄O₇ NPs

Both H₂O₂ and hydroxyl radicals have strong oxidizing ability and can oxidize biological macromolecules, such as proteins and phospholipids [32]. Our study found that Tb₄O₇ NPs with oxidase-like activity can catalyze the production of H₂O₂ and further produce hydroxyl radicals. Therefore, the oxidase-like activity of the Tb₄O₇ NPs makes them potentially useful as antibacterial agents. We evaluated the antibacterial activity of Tb₄O₇ NPs against E. coli and S. aureus. A colony-forming units plate counting method was used to determine the antibacterial ability (Fig. 3a, b). As compared to the PBS control group, Tb₄O₇ NPs exhibited potent antimicrobial activity against both S. aureus and E. coli in a concentration-dependent manner. At a concentration of 25 μg/mL, Tb₄O₇ NPs exhibited only modest antibacterial effects against S. aureus; more than 80% of the bacterial cells survived. However, when the concentration of Tb₄O₇ NPs was increased to 100 μg/mL, nearly 90% of the S. aureus were killed. A similar trend of antibacterial effects were observed towards the E. coli.

Fig. 3.

The effect of Tb₄O₇ NPs on survival rates of bacteria a CFUs of S. aureus following incubation with Tb₄O₇ NPs; b CFUs of E. coli following incubation with Tb₄O₇ NPs; c Representative fluorescence and SEM images of S. aureus after Tb₄O₇ NPs treatments. Bacterial cells were treated with 1) PBS as control, 2) 25 μg/mL Tb₄O₇ NPs, 3) 50 μg/mL Tb₄O₇ NPs or 4) 100 μg/mL Tb₄O₇ NPs; d Representative fluorescence and SEM images of E. coli after Tb₄O₇ NPs treatments. Bacterial cells were treated with 1) PBS as control, 2) 10 μg/mL Tb₄O₇ NPs, 3) 25 μg/mL Tb₄O₇ NPs or 4) 50 μg/mL Tb₄O₇ NPs. **p < 0.01 and ***p < 0.001 vs control

To further investigate the interaction between Tb₄O₇ NPs and bacteria, a fluorescent-based cell live/dead assay was conducted. As shown in Fig. 3c, d, Tb₄O₇ NPs exhibited significant antibacterial activity against both S. aureus and E. coli, which was consistent with the aforementioned results. The exposure of S. aureus cells to Tb₄O₇ NPs at a concentration of 100 μg/mL resulted in nearly 100% lethality, as evidenced by the dominant red fluorescent signal. At a concentration of 50 μg/mL, Tb₄O₇ NPs completely inhibited the bacterial growth of E. coli.

The morphology and membrane integrity of bacteria were then determined by SEM (Fig. 3c, d). Untreated S. aureus displayed a typical rod-shaped structure with a continuous, smooth surface. When exposure to the 50 μg/mL of Tb₄O₇ NPs, the S. aureus bacterial cell walls became partially wrinkled and discontinuance. Notably, after treatment with 100 μg/mL of Tb₄O₇ NPs, the S. aureus bacterial cell walls showed much more pronounced damage, indicating stronger antibacterial effects at higher concentrations of Tb₄O₇ NPs. A similar tendency was found for E. coli. The loss of membrane integrity of E. coli was observed at concentrations lower than 100 μg/mL of Tb₄O₇ NPs treatment. Moreover, Tb₄O₇ NPs were observed by SEM and SEM-energy dispersive X-ray spectroscopy (EDS) to aggregate on the surfaces of S. aureus and E. coli (Additional file 1: Figure S4).

It is known that the proton motive force decreases the local pH (as low as pH 3.0) in the cytoplasm and membrane of bacteria cells [33, 34]. Since we found that Tb₄O₇ NPs exhibit oxidase-like activity under acidic conditions, we speculate that the antibacterial mechanism of Tb₄O₇ NPs may arise from their oxidase activity to accelerate the process of bacterial cell oxidation and consumption of antioxidant biomolecules, leading to a reduction of oxygen products including H₂O₂ along with other antibacterial activity from the accumulation of ROS. To confirm this hypothesis, the intracellular levels of ROS were determined using the florescent probe, DCFH-DA (Fig. 4). For both S. aureus and E. coli, the untreated cells showed extremely weak fluorescence, indicating low levels in the formation of intracellular ROS. In contrast, bacterial cells exposed to Tb₄O₇ NPs showed high levels of ROS formed within the cellular cytoplasm, as evidenced by the strong fluorescence signal. We also found that the generation of ROS is Tb₄O₇ NPs dose-dependent (Additional file 1: Figure S5).

Fig. 4.

a Fluorescence images of bacterial cells. b Analysis of the ROS levels by microplate reader. ***p < 0.001 vs control

In vivo wound disinfection effect of Tb₄O₇ NPs

The above findings suggest that Tb₄O₇ NPs may have role as an antibacterial agent for bacterial infections in vivo. To assess the antibacterial capacity of Tb₄O₇ NPs in vivo, a wound infection model was constructed using BALB/c mice. A wound was introduced on the back of the mouse and an infection was established by implanting S. aureus into the wounded area. After infection was established, PBS or Tb₄O₇ NPs were applied to the infected wound. Figure 5a, b shows the progress of the wounds. Compared with the control (PBS treatment) group after 3 days, the wound area was reduced under Tb₄O₇ NPs treatment. After treating of 7 days with Tb₄O₇ NPs treatment, the wounds were nearly healed completely. In contrast, obvious scab was observed from the control group, indicating incomplete recovery.

Fig. 5.

a Photographs of wounds on the backs of mice in control (PBS) and Tb₄O₇ NPs treatment groups (n = 5). Scale bar: 5 mm. b Related wound size in each treatment group. c Bacterial number of infected wounds on the 7th day. ***p < 0.001 vs control

Biosafety of Tb₄O₇ NPs

Biosafety is an important factor for antimicrobial agents designers. To assess the biosafety of Tb₄O₇ NPs, we first determined the effects of Tb₄O₇ NPs on red blood cells and HUVECs in vitro. The effect of Tb₄O₇ NPs on cell membrane disrupt was first determined by a red blood cell hemolysis assay. As shown in Fig. 6a, pure water can cause severe red blood cells hemolysis within 2 h. In contrast, the introduction of Tb₄O₇ NPs did not cause signs of hemolysis. In this experimental result, the hemolysis rate was still less than 1% at a concentration of 100 µg/mL, which fully demonstrated that Tb₄O₇ NPs have good blood compatibility. Meanwhile, the cytotoxicity tests on mammalian cells HUVECs further confirm the biosafety of Tb₄O₇ NPs (Fig. 6b).

Fig. 6.

a The hemolysis ratio of red blood cells. The insert images of tubes containing red blood cells solution show the direct observation of hemolysis. Tube 1: PBS buffer; Tube 2–5: 25, 50, 100, and 200 μg/mL Tb₄O₇ NPs; Tube 6: ultrapure water. b MTT assays determined cell viability of HUVECs after Tb₄O₇ NPs treatment

Then, the biosafety of Tb₄O₇ NPs in vivo were determined. As shown in Fig. 7a, the indicators in blood were within the normal range. The major mouse organs (heart, liver, spleen, lung, and kidney) were formalin-fixed and processed for the evaluation of H&E sections by histopathology (Fig. 7b). No obvious mouse organ damage was observed from Tb₄O₇ NPs treatment.

Fig. 7.

In vivo toxicity of Tb₄O₇ NPs. a The blood biochemistry data of the mice treated with Tb₄O₇ NPs after 7 d (n = 5). b Histological data (H&E staining images) are obtained from the major organs of mice treated with Tb₄O₇ NPs after 7 d. Scale bar = 100 μm

Conclusions

In summary, this study demonstrates for the first time that Tb₄O₇ NPs exhibit oxidase-like activity. In addition, the results of this study established a relationship between the oxidase-like enzyme activity of Tb₄O₇ NPs and their antibacterial properties. The data collected from this work revealed that the oxidase-like activity of Tb₄O₇ NPs was able to function with a variety of substrates, including biomolecules, and resulted in the generation of ROS, which further enhanced their antibacterial activity. The application of the antibacterial activities of Tb₄O₇ NPs were demonstrated in a wound infection mouse model. Our study provides evidence that Tb₄O₇ NPs can be utilized as an efficient antibacterial agent and the potential applications in wound healing are promising.

Abbreviations

Tb₄O₇

terbium oxide

NPs

nanoparticles

H₂O₂

hydrogen peroxide

ROS

reactive oxygen species

ABTS

diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)

OPD

o-phenylenediamine

AA

ascorbic acid

DCFH-DA

2′,7′-dichlorodihydrofluorescein diacetate

BMPO

5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide

CCK-8

Cell Counting Kit-8

E. coli

Escherichia coli

S. aureus

Staphylococcus aureus subsp. aureus

HUVEC

human umbilical vein endothelial cells

TEM

transmission electron microscopy

ESR

electron spin resonance

CFUs

colony forming units

PI

propidium iodide

SEM

scanning electron microscope

DLS

dynamic light scattering

EDS

energy dispersive X-ray spectroscopy