Biocompatible tellurium nanoneedles with long-term stable antibacterial activity for accelerated wound healing.

Tellurium (Te) nanomaterials (NMs) have emerged as a new antibacterial candidate to respond to the complex global health challenge of bacterial resistance. Herein, Te nanoneedles (NNs) that act both chemically and physically on bacteria are synthesized by a facile method using Na2TeO3, urea and glucose. It is found that the prepared Te NNs have a strong affinity to the cell membrane of bacteria and subsequently promote the generation of reactive oxygen species (ROS) in bacteria, resulting in an excellent antibacterial effect toward Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). What's more, this needle-like morphology also can physically damage the bacterial cell membranes. The Te NNs per se are inert in mammalian cells to produce ROS at a proper concentration, indicating considerable biocompatibility of this material. As a proof-of-concept, the antibacterial Te NNs were used as an anti-inflammatory reagent for promoting bacteria-infected wound healing in vivo, during which Te NNs caused no evident side effects to major organs in mice. Additionally, the antibacterial activity is maintained in the presence of surface oxidation of Te NNs after long-term dispersion in phosphate buffered saline solution. The needle-like Te NMs with long-term antibacterial stability and good biocompatibility have great potential for the treatment of associated infectious diseases.

Introduction

Clinical operations such as prostheses replacement, dental operations, and open wounds have great risks in bacterial infections [[1], [2], [3], [4], [5]]. Although various antibiotics have been developed for solving these tough infections, the abuse of broad-spectrum antibiotics causes ever-increasing drug-resistance bacteria, which is currently one of the most daunting healthy crises for modern antibacterial medicine [6,7].

The merits of large surface-to-volume ratio, special morphology, low toxicity and facile surface functionalization ability confer nanomaterials (NMs) as rising stars for biomedical application, especially in the antibacterial field [8]. Various inorganic nanomaterials, such as Ag-based nanoparticles (NPs), Cu-based NPs, TiO2 NMs, ZnO NMs and black/red phosphorus have been developed for anti-pathogenic microorganisms [[9], [10], [11], [12], [13], [14], [15], [16], [17]]. Those NPs could produce abundant reactive oxygen species (ROS) for bacterial killing by themselves or under external stimuli (laser, magnetic, light). Although the ROS make a great contribution to sterilization, it also poses a great threat to cells and tissues and will induce inflammatory responses during the therapeutic process [18,19]. To promote wound healing and kill bacteria at the same time, 2D materials such as black/red phosphorus nanosheets [15,17], Ti3C2Tx MXene [20], graphitic carbon nitride-based materials [[21], [22], [23]] and metal-organic framework-based materials [24] have been developed recently. However, the synthesis of these nanomaterials is generally complicated, with low synthesis efficiency and high cost. Moreover, the need of external stimuli also limits the convenience of the application.

Elementary substance Te as a p-type semiconductor with band-gap energy of 0.35 ​eV, whose toxicity is much lower than other Te compounds, is recently recognized as a competitive candidate that can be used as antibacterial agents [[25], [26], [27]]. However, there has been no consensus on the antibacterial mechanism of the Te NPs. And notably, the tricky biofilms formed by bacteria, which support a more comfortable environment for bacterial attachment and growth and then become a “protective umbrella” for bacteria, are a core reason for repeated infection [28,29]. The biofilm destruction ability of Te NMs has rarely been explored. In addition, the variation in the antibacterial properties of Te NPs with the oxidation is well worthy of exploration since the Te NPs are easily oxidized in air as well as in solution [30,31].

In this work, we develop a facile one-step approach for the efficient synthesis of Te nanoneedles (NNs). It is found that the Te NNs can efficiently attach to the surface of S. aureus and E. coli to directly promote the generation of ROS in bacteria without external excitation required, which causes the irreversible destruction of membrane structure and deoxyribose nucleic acid (DNA), leading to the death of the bacterial cells. Moreover, the Te NPs are provided with needle-like structure that can puncture bacterial membranes and then cause physical damage. The prepared Te NNs exhibit retained chemical and physical damage to bacteria during long-term exposure of 25 days. Noteworthy, the Te NNs themselves are inert to produce ROS and will not stimulate mammalian cells to generate ROS at proper doses, resulting in excellent biocompatibility. Interestingly, Te NNs or surface oxidized Te NNs (Te–SO NNs) exhibit similar antibacterial activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). We further explore the antibacterial ability and wound healing efficiency of Te NNs in vivo, the results show that they can effectively eliminate bacteria and significantly accelerate healing of S. aureus-infected and S. aureus & E. coli co-infected wounds. Therefore, these biocompatible Te NNs could work as a promising candidate to kill bacteria for the treatment of associated infectious diseases.

Experiments

Preparation and characterization of Te NNs and Te–SO NNs

The Te NNs were synthesized via a facile one-step method by reacting Na2TeO3, urea and glucose (analytical grade, Aladdin, Shanghai, China) at a ratio of 1:10:10 for 1 ​h at 80 ​°C [[32], [33], [34], [35], [36]]. Briefly, Na2TeO3 (10 ​mM, 50 ​mL) was dissolved in deionized water and stirred in a magnetic mixer at 80 ​°C, set as A solution. The mixture of urea and glucose (100 ​mM, 50 ​mL) was dissolved in deionized water, set as B solution. Then, B was added to A solution, after stirring for 15 ​min, NaOH (2 ​M, Aladdin, Shanghai, China) was subsequently added dropwise to adjust the pH value ∼9. The mixed solution reacted under vigorous magnetic stirring at 80 ​°C for 1 ​h. After that, the container was cooled to room temperature naturally. The Te NNs synthesized above were washed several times with deionized water and ethanol. Finally, the products were dried in vacuum at 60 ​°C for characterization. Furthermore, the Te NNs were dispersed in the phosphate buffered solution (PBS, Sigma-Aldrich) for 14 days to form Te–SO NNs, and the products were also dried in vacuum at 60 ​°C for characterization.

The prepared Te and Te–SO NNs were characterized using a series of methods including HRTEM, XRD, Raman and XPS, etc. The NNs suspension was placed on a carbon-coated copper grid and observed using HRTEM (JEOL 2100, Japan). The X-ray diffraction (XRD) patterns of Te and Te–SO NNs were acquired using a Multi-position automatic sampling X-ray diffractometer (X'pert Powder, PANalytical) in the 2-theta range of 5°–90° under 40 ​kV and 30 ​mA, with λ ​= ​1.5406 ​Å radiation. Raman spectra of Te and Te–SO NNs were recorded on a confocal Raman microscope (HJY LabRAM Aramis, France) with a 532 ​nm laser source at an output power of ∼5 ​mW for excitation. The X-ray photoelectron spectra (XPS) of Te and Te–SO NNs were taken on a ThermoFisher ESCALAB Xi+ with an incident monochromated X-ray beam from the Al target.

Cell culture and cytotoxicity in vitro

Murine fibroblast cells (L929) were selected to evaluate the cytotoxicity of the Te and Te–SO NNs by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. L929 cells (100 ​μL) were seeded into a respective 96-well plate at a density of 1 ​× ​105 ​cells·cm−2 and cultured with Dulbecco's modification of Eagle's medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (P/S). After the Te NNs were suspended in the PBS, gradient concentrations of 200, 100, 50, 25, 12.5, 6.25 and 0 ​μg ​mL−1 were added to the plates and co-cultured at 37 ​°C for 24 ​h. Then, cell proliferation was assessed by the MTT assay at 37 ​°C for 4 ​h. The formazan was then dissolved using dimethyl sulfoxide (DMSO, sigma, USA), and the optical density (OD) was measured at 490 ​nm using a microplate reader (Infinite 200 Pro). A certain concentration of Te NNs was dispersed in PBS for 14 ​d and then repeat the operation above to evaluate the cytotoxicity of the Te–SO NNs.

In addition, the visualized viability of the L929 ​cells was determined with a live/dead staining under the fluorescence microscope. The cells were cultured with the NNs (100 ​μg ​mL−1) in 24-well plate for 24 ​h. At the end of the incubation period, the medium was removed and 250 ​μL Calcein-AM/PI staining solution (2 ​μM Calcein-AM and 4.5 ​μM PI in DMEM) was added into 24-well plate, then cultured for 20 ​min at 37 ​°C in darkness. Finally, the cells were washed gently with PBS at least three times and placed under a fluorescence microscope to record the cell growth on the Te NNs and Te–SO NNs.

In vitro antibacterial activity

The S. aureus and MRSA were grown in sterilized Nutrient-Broth (NB) medium (0.3% w/v beef extract, 0.5% w/v NaCl, 1% w/v tryptone). And the E. coli were grown in sterilized Luria-Bertani (LB) medium (0.5% w/v yeast extract, 0.5% w/v NaCl, 1% w/v tryptone) by using a shaking incubator at 150 ​rpm at 37 ​°C overnight. The bacterial concentration was defined by measuring the OD value at 600 ​nm, and OD600 ​= ​1 was considered 5 ​× ​108 ​CFU ​mL−1. In the control group, the bacteria S. aureus (or MRSA) were adjusted to a concentration of 2 ​× ​106 ​CFU ​mL−1 in PBS containing 3% NB and E. coli were diluted to the same concentration in PBS containing 1% LB. The concentrations of Te and Te–SO NNs (as well as Te NNs dispersed in PBS for 6 months) were varied from 0 to 100 ​μg ​mL−1 and then introduced to bacterial suspension. The resultant suspension was incubated in a biochemical incubator at 37 ​°C for 24 ​h. The bacteria culture suspension was diluted 1000 times in PBS and a suspension of 100 ​μL from each tube was uniformly spread on the NB agar plates and the number of viable bacterial colonies was counted after incubation at 37 ​°C for 24 ​h. To quantify the antibacterial ability, the bactericidal rate was calculated based on the following equation:Where Ncontrol is the average number of bacteria in the control sample (CFU/sample), and NNNs is the average number of bacteria in the testing samples (CFU/sample). The minimum inhibitory concentration (MIC) is set as the lowest drug concentration that can inhibit the growth of 80% of pathogenic bacteria after 24 ​h of in vitro culture. Furthermore, the visualized viability of the bacteria treated with 100 ​μg ​mL−1 Te and Te–SO NNs was determined with SYTO 9/PI staining (SYTO 9 and PI, Thermo Fisher Scientific Co, Ltd, USA) at 37 ​°C for 30 ​min and then observed under confocal laser scanning microscope (CLSM).

To directly observe the morphology changes of bacteria after treated with Te and Te–SO NNs, scanning electron microscopy (SEM, ZEISS, Germany) was employed. After different treatments (as described above) and co-cultured for 24 ​h, the samples were gently rinsed with PBS and fixed with 3% glutaraldehyde overnight. Then, the samples were subsequently dehydrated with 20, 40, 60, 80, 90, 95and 100% ethanol. The samples were dried naturally and coated with gold for SEM observation.

In vitro biofilm formation and antibiofilm ability to E. coli

For the formation of the biofilm, 1 ​mL of E. coli suspensions with OD600 ​= ​1 were seeded on the surface of the sterilized Si plates and incubated in a 24-well plate at 37 ​°C. To further observe the antibiofilm ability of Te and Te–SO NNs, 100 ​μg ​mL−1 Te and Te–SO NNs were added to the bacterial suspension above. After 24 ​h of incubation, the Si plates were gently washed three times with PBS to remove the suspended bacteria and fixed with 3% glutaraldehyde overnight. Then, the samples were subsequently dehydrated with 20, 40, 60, 80, 90, 95 and 100% ethanol. Finally, the samples were dried naturally and coated with gold for SEM observation. In addition, the E. coli were seeded on the surface of the sterilized glass slides to form the biofilm as above. After E. coli biofilm co-cultured with Te and Te–SO NNs (100 ​μg ​mL−1) for 2 ​h and 12 ​h, samples were gently rinsed twice with 1 ​mL of PBS to remove non-adherent planktonic bacteria. Samples were then incubated in 500 ​μL of 0.4% MTT solution at 37 ​°C for 3 ​h followed by dissolution of the purple formazan crystals in 500 ​μL of DMSO. Absorbance of the purple colored solution was measured at 490 ​nm using a microplate reader (Infinite 200 Pro).

Assessments of AMR evolution in bacteria

On the basis of the operation and results about in vitro antibacterial activity, 2 ​× ​106 ​CFU ​mL−1 bacteria (S.aureus and E. coli) were inoculated into PBS containing 3% NB with dosages of 0.25 ​× ​MIC, 0.5 ​× ​MIC, 1 ​× ​MIC, 2 ​× ​MIC, 4 ​× ​MIC, 8 ​× ​MIC Te NNs. Next, after incubated for 24 ​h at 37 ​°C, it was spread on a nutrient plate to evaluate the sterilization effect. Then, the colonies of S. aureus and E. coli with a certain inhibitory effect on the growth but could ensure the basic growth of the bacteria were collected in culture and shaken for 4 ​h at 37 ​°C, and subsequently, repeat the above-mentioned cultivation operation. This continuous passage was repeated every day for 25 days.

Binding ability

Isothermal titration calorimetry (ITC) experiments were conducted with a MicroCal VP-ITC instrument at 25 ​°C. Lipopolysaccharide (LPS) solution, Peptidoglycan (PG) solution and the Te NNs solution were prepared in PBS. For a typical titration, 10 ​μL LPS or PG solution was injected into the Te NNs solution within a duration of 20 ​s. The spacing time between two continuous injections was 2 ​min. The heat for each injection was determined by the integration of the peak area in the thermogram corresponding with the reaction time.

Intracellular ROS generation

The S. aureus and E. coli suspension (2 ​× ​106 ​CFU ​mL−1) were given different treatments as described above. The 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) stock solution (10 ​mM) was diluted with PBS to obtain the working solution (20 ​μM). The bacterial culture along with NNs suspension (100 ​μg ​mL−1 in PBS) were incubated with the DCFH-DA working solution at 37 ​°C for an additional 1 ​h, and the fluorescence intensity of samples was observed by fluorescence microscopy and detected by a microplate reader (Infinite 200 Pro) at 525 ​nm under the excitation of 488 ​nm.

The intracellular ROS production of L929 ​cells was also detected by DCFH-DA. In brief, L929 ​cells were seeded in culture dishes (1 ​× ​105 ​cells·mL−1) and cultured for 12 ​h. After washing with PBS three times, the cells along with DCFH-DA probe were incubated for 2 ​h with/without NNs (100 ​μg ​mL−1). Finally, the cells were washed with PBS and observed under CLSM.

Bacterial genomic DNA integrity

After the bacteria with/without the treatment of NNs (100 ​μg ​mL−1) were cultured for 24 ​h, genomic DNA was extracted using Bacteria DNA Kit (Solarbio, Beijing, China). The concentration and purity of the bacterial DNA were detected by Nano-Drop 2000 (Thermo Fisher Scientific, USA). Agarose gel was prepared with 1 ​× ​TAE buffer, and ethidium bromide (5 ​μL per 100 ​mL of the gel) was used to monitor the size of the analyzed fragment. Electrophoresis (Bio-Rad, Hercules, CA, USA) was run at 110 ​V for 30 ​min. The bands were visualized by a UV transilluminator (Bio-Rad, GDS-7500, USA).

In vivo anti-infection and wound healing

The permission for the animal experiments was obtained from the Institutional Animal Care and Use Committee (IACUC) of General Hospital of Southern Theater Command of PLA. Male Sprague-Dawley (SD) rats (∼250–300 ​g) were purchased from Medical Experimental Animal Center of Guangdong Province. After being anesthetized with 3% pentobarbital at a dose of 45 ​mg ​kg−1 of body weight, the fur on the dorsum skin of the rat was shaved off, and the skin was sterilized using 0.5% iodophor for aseptic surgery. To establish the rat model of infectious wound, two full-thickness skin wounds (diameter≈15 ​mm) were excised using a sterile round cutaneous punch on the dorsal side skin. 10% (wt/v) polyvinyl alcohol (PVA) was dissolved in PBS and stirred in a magnetic mixer at 90 ​°C for 4 ​h to prepare PVA hydrogels. And the Te NNs suspension was mixed in PVA hydrogels to further form the Te NNs/PVA hydrogels (the concentration of Te NNs is 100 ​μg ​mL−1). The rat model of infectious wound was divided into two series including S. aureus infected model and S. aureus & E. coli co-infected model. For S. aureus infected series, an S. aureus suspension (100 ​μL, 5 ​× ​108 ​CFU ​mL−1) was directly dropped on the wound and the wounds were covered with sterile gauze for 24 ​h. A total of 15 rats were randomly divided into three groups (5 rats per group): control group (treated with 100 ​μL PBS), PVA group (treated with 100 ​μL PVA hydrogel), and Te NNs/PVA group (treated with 100 ​μL Te NNs/PVA hydrogel). As to S. aureus and E. coli co-infected series, the procedure is the same as that of the S. aureus series apart from different bacterial suspension addition, in which an S. aureus and E. coli mixture (100 ​μL, 5 ​× ​108 ​CFU ​mL−1) was dropped on the wound instead of S. aureus suspension. Photographs of the wound on the mice during the wound healing process (day 0, 4, 7, 10, and 14) were recorded using a digital camera. And the wound area was quantified using image analysis software (Image J). The wound healing rate was calculated using the following equation:Where Ainitial is the initial wound area (day 0), and Atime is the wound area at different time points.

In addition, bacteria were collected from the wounds and suspended in 1 ​mL PBS at days 4, 7, 10, and 14. The samples were diluted 10 times and 100 ​μL of the diluted solutions were spread on NB agar plates and incubated for 24 ​h at 37 ​°C. Then the number of bacterial colonies on agar plates was calculated using Image J software.

Histological analysis

Hematoxylin & eosin (H&E) staining and Masson's trichrome staining of the wound tissue after 7 and 14 days of treatment were performed to assess the wound recovery. The rats were sacrificed with an overdose of pentobarbital, after that the wound tissues with adjacent normal skin were harvested and fixed in the 4% paraformaldehyde solution for 24 ​h at 4 ​°C. After 14 days of different treatments, the major organs (heart, liver, spleen, lung, and kidney) were also collected and fixed for H&E staining.

Statistical evaluation

All quantitative tests were carried out at least in triplicate, and the data were expressed as mean ​± ​standard deviation (SD). The statistical comparison was tested by Tukey's multiple comparisons test using SPSS (version 10; SPSS Inc, Chicago, IL). The value with ∗p ​< ​0.05 were considered statistically significant, and ∗∗p ​< ​0.01 were set as highly statically significant.

Results and discussion

Characterization

The TEM image in Fig. 1a shows the needle stick-like Te NNs with a diameter of ∼40 ​nm, and lengths range from ∼500 ​nm to even 1 ​μm. The electron diffraction pattern (inset of Fig. 1a) and HRTEM image (Fig. 1b) present the crystalline structure of the Te NNs with interplanar spacing d ​= ​0.3218 ​nm, which is consistent with (101) plane of hexagonal tellurium crystal. Interestingly, the surface of Te NNs was oxidized after preserved in the PBS for 14 ​d, namely Te–SO with increased diameter to 100 ​nm (Fig. 1c). Fig. 1d illustrates the X-ray diffraction (XRD) patterns of the Te and Te–SO NNs, all peaks in the pattern of Te samples are in good agreement with the standard literature data (JCPDS: 36–1452). Notably, some peaks in Te–SO NN samples are attributed to orthorhombic TeO2 (JCPDS: 74–1131) have appeared (star mark), which indicates that part of Te NNs are oxidized and therefore Te–SO NNs are formed. The Raman scattering spectra taken for the Te NNs and Te–SO NNs are depicted in Fig. 1e. The characteristic vibration peaks at 121.7 ​cm−1, 141.1 ​cm−1 are observed in both Te and Te–SO samples, which are close to those reported Te NPs previously [[37], [38], [39]]. Moreover, the peaks at 180 ​cm−1, 226 ​cm−1, 444 ​cm−1, 583 ​cm−1, 650 ​cm−1 and 665 ​cm−1 which are attributed to β-TeO2 [40] emerge in the Te–SO samples (star mark) due to surface oxidation, which is consistent with the XRD results above. Additionally, the characteristic XPS spectra (Te 3d5/2 and Te 3d3/2 peaks) further support the formation of Te and Te–SO NNs. In these XPS region spectra (Fig. 1f), the Te 3d5/2 and Te 3d3/2 peaks at 573.4 ​eV and 583.7 ​eV associated with Te (0) appear in the Te samples [38,41]. While the appearance of additional Te 3d5/2 and Te 3d3/2 peaks at 576.4 ​eV and 586.6 ​eV which arise from Te (Ⅳ) is observed in the Te–SO samples [[42], [43], [44]].

Fig. 1

Characterization of the Te and Te–SO NNs. (a) TEM image of the Te NNs; inset: Selected area electron diffraction (SAED) images. (b) HRTEM image of the Te NNs; the lattice spacing corresponds to (101) in XRD. (c) TEM image of the Te–SO NNs. (d) XRD pattern of the Te and Te–SO NNs. (e) The Raman spectra and (f) XPS spectra of the Te and Te–SO NNs.

In vitro cytotoxicity assessment

Cytotoxicity is the biggest concern in biomedical applications of biomaterials [45]. A proper dose of Te NNs can kill bacteria but not do harm to mammalian cells selected here. As shown in Fig. 2a, the cell viability is slightly lower than 80% when the concentration of Te or Te–SO NNs is up to 200 ​μg·mL−1. When their concentrations are lower than 100 ​μg ​mL−1, both Te NNs and Te–SO NNs have a negligible effect on the L929 ​cells. Subsequently, the L929 ​cells were stained with calcein-AM and propidium iodide (PI) to identify live and dead cells, respectively. The full-screen green fluorescence in the control group as well as NNs groups indicates the excellent biocompatibility of Te and Te–SO NNs (100 ​μg ​mL−1). Furthermore, The ROS production in L929 ​cells is monitored using a green probe, DCFH-DA. As shown in Fig. 2c, L929 ​cells internalizing Te or Te–SO NNs exhibit negligible fluorescence along with the control group, illustrating the inert character of NNs, which further confirms their biocompatibility.

Fig. 2

Cytotoxicity of the Te and Te–SO NNs. (a) Cytotoxicity of L929 co-cultured with different concentrations of Te and Te–SO NNs; (b) Fluorescence images of the L929 ​cells after Live/Dead staining in the different groups; (c) Bright-field images and DCFH-DA fluorescence images of L929 ​cells with several treatments for detection of ROS level.

Antibacterial activity to S. aureus

We further explored the antibacterial ability of Te NNs and Te–SO NNs toward gram-positive bacteria (S. aureus). As shown in Fig. 3, Te NNs can kill all S. aureus at the concentration of 100 ​μg ​mL−1 and Te–SO NNs also exhibit excellent antibacterial properties toward S. aureus at the same concentration with an antibacterial rate of 98.2% (Fig. 3a and b). Nevertheless, when their concentration falls to 50 ​μg ​mL−1, dozens of colonies can be observed on Te NNs as well as Te–SO NNs group (Fig. 3a), which indicates that the antibacterial ability is concentration-dependent. When the concentration further decrease to 25 ​μg ​mL−1, Te NNs exhibit poor bactericidal effect. What's more, the antibacterial activity attenuates when oxidation occurs on the surface of Te NNs, which decreases from 87.6 to 78.2% at the concentration of 50 ​μg ​mL−1. We set the lowest drug concentration that can inhibit the growth of 80% of pathogenic bacteria after 24 ​h of in vitro culture as the minimum inhibitory concentration (MIC). Therefore, the MIC of Te and Te–SO NNs to S. aureus are listed as 50 and 75 ​μg ​mL−1, respectively. After a longer dispersion of 6 months, partially oxidized Te NNs also show bactericidal activity to S. aureus with the antibacterial rate of 100% at the concentration of 100 ​μg ​mL−1, and MIC is listed at 12.5 ​μg ​mL−1 (Figure S1). Furthermore, as the treatment of multidrug-resistant bacteria is still an intractable health challenge, the antibacterial ability of Te and Te–SO NNs to one of the most serious superbugs—methicillinresistant Staphylococcus aureus (MRSA) is observed in Figure S2. Unfortunately, both Te and Te–SO NNs perform inferiorly in MRSA elimination even at the concentration of 100 ​μg ​mL−1, with the antibacterial rate of only respective 72.27% and 66.91% (Figure S2a,b).

Fig. 3

Antibacterial activity of Te and Te–SO NNs toward S. aureus. (a) Variable numbers of colonies and (b) antibacterial rate in the presence of Te and Te–SO NNs at different concentrations. (c) SYTO 9/PI staining of S. aureus treated with Te and Te–SO NNs (100 ​μg ​mL−1). (d) SEM images of S. aureus with the treatment of Te or Te–SO NNs (100 ​μg ​mL−1).

Considering the immediate and long-term antibacterial efficiency together, we choose 100 ​μg ​mL−1 Te NNs for further experiments and the effects of Te–SO NNs at the same concentration are simultaneously investigated. The S. aureus killing effect of NNs is further observed using bacterial SYTO 9/PI staining. SYTO 9 is a membrane-permeable dye that can enter all bacterial cells [28,29], whereas PI is excluded from cells with structurally intact cytoplasmic membranes, which only penetrates dead bacterial cells with disrupted membranes. These two dyes provide a rapid and reliable method for discriminating between live and dead bacteria. As performed in Fig. 3c, all bacteria are alive (stained by SYTO 9, green fluorescence) in the control group, while most of bacteria are dead (staining by PI, red fluorescence) after 24 ​h treatment of Te NNs and Te–SO NNs. This confirms the excellent S. aureus disinfection of Te NNs as well as Te–SO NNs. In addition, the morphology changes of S. aureus are visualized through SEM (Fig. 3d). The spherical well-dividing bacterial cells with intact membrane structure and smooth surface are observed in the control group. In comparison, the bacteria treated with Te and Te–SO NNs are shrunk and their membranes become incomplete, sunken, and even melted (orange arrow), which results in the leakage of cytoplasm and thereby causes the death of bacteria. Generally, antimicrobial resistance (AMR) will cause increased threat in clinic, hence the possible induced resistance of bacteria with long-term exposure to NNs is assessed. The results reveal that the prepared Te NNs exhibit retained antibacterial effects to the S. aureus during 25-day exposure (Figure S3).

Antibacterial and antibiofilm activity to E. coli

The antibacterial activity of NNs toward Gram-negative bacteria (E. coli) is illustrated in Fig. 4. The viable cell counting method is used to evaluate E. coli elimination ability of Te and Te–SO NNs (Fig. 4a). The results demonstrate that Te and Te–SO NNs can kill all E. coli even at the concentration of 50 ​μg ​mL−1 (Fig. 4a and b), illustrating a stronger antibacterial effect than against S. aureus. What's more, both Te and Te–SO NNs maintain excellent antibacterial efficiency to E. coli until the concentration drops to 1.5625 ​μg ​mL−1. And as shown in Figure S4, the antibacterial efficiency of Te NNs also maintain at an ideal level with MIC of 3.125 ​μg ​mL−1 when dispersed in PBS for 6 months. Furthermore, Te NNs perform no detectable resistance after the serial incubation with the selected E. coli at sub-MIC over a 25-day period (Figure S5).

Fig. 4

Antibacterial and antibiofilm activity of Te and Te–SO NNs toward E. coli. (a) Variable numbers of colonies and (b) antibacterial rate in the presence of Te and Te–SO NNs at different concentrations. (c) SYTO 9/PI staining of E. coli treated with Te and Te–SO NNs (100 ​μg ​mL−1). SEM images of (d) E. coli and (e) E. coli biofilms with the treatment of Te or Te–SO NNs (100 ​μg ​mL−1).

The SYTO 9/PI staining shown in Fig. 4c further confirms their bactericidal activity, in which the full-screen green is covered by red in 24 ​h of contact with 100 ​μg ​mL−1 NNs. The morphological change in E. coli cells caused by NNs is examined by SEM (Fig. 4d). The rod-shaped cells with intact cell membranes are investigated in the control group. However, when treated with Te and Te–SO NNs, a severe folding and shrinking of bacterial cytoplasm can be observed.

More importantly, E. coli can easily adhere to the surface of implants, then form mature and stable biofilms, which can prevent the penetration of both antibiotics and immune cells, leading to difficulty in bacteria eradication. E. coli biofilms are certainly the major cause of morbidity and mortality of bacterial infections [46,47]. To visualize the antibiofilm capability toward E. coli of the Te and Te–SO NNs, the E. coli biofilms treated with NNs are investigated. The SEM images in Fig. 4e show that bacterial biofilms in the control group are distributed evenly and grow densely. By contrast, the E. coli biofilms are significantly destructed with obvious changes of bacterial morphology in the presence of Te and Te–SO NNs, indicating excellent antibiofilm activity of the NNs to E. coli (Fig. 4e). Furthermore, the quantitative assessment for E. coli biofilm growth by MTT assay (Figure S8) shows that both Te and Te–SO NNs have significantly disrupted biofilm at the concentration of 100 ​μg ​mL−1 as compared to control group, which comfirms the outstanding antibiofilm activity of the NNs. The Te NNs and Te–SO NNs exhibit excellent antibiofilm activity in a relatively short period of 2 ​h with biofilm viability of 52.7% and 51.1%, respectively. When the treatment time is extended to 12 ​h, the biofilm viability is further reduced to respective 27.1% and 28.3%.

Antibacterial mechanism

The antibacterial activity discussed above shows the varying degrees of morphological damage caused by Te and Te–SO NNs. In general, the Gram-positive bacteria possess thick peptidoglycan (PG) layer and the lipopolysaccharide (LPS) accounts for a large portion in Gram-negative bacterial cell walls [48,49]. To further investigate the interaction process of Te NNs to PG and LPS, isothermal titration calorimetry (ITC) is applied and the resulting isotherm was fitted to a one-site binding model using the nonlinear Levenberg-Marquardt fitting algorithm by MicroCal PEAQ-ITC Analysis Software (Fig. 5a and b). The value of ΔH and ΔS is employed to determine the driving force during the interaction process which includes the van der Waals force, hydrophobic interactions and hydrogen bonds [50,51]. The ΔH and -TΔS of Te NNs to LPS and PG are almost the same, implying that the main driving force is similar in these two interaction processes. Moreover, the equilibrium dissociation constants (KD) of Te NNs to PG and LPS are respectively listed as 37.8e−6 and 31.1e−6, suggesting that Te NNs have a great affinity to PG as well as LPS. However, the ITC result of Te–SO NNs to PG performs insignificant endothermic or exothermic behavior (Figure S6a), which indicates that this binding ability attenuates with surface oxidation of Te NNs, therefore reducing the bactericidal ability to S. aureus. And the binding ability of Te and Te–SO NNs to PG is also confirmed using a PG kit. After the mixture of NNs with PG is shaken for 4 ​h at 4 ​°C, the binding rate of Te NNs to PG is nearly twice that of Te–SO NNs (Figure S6b).

Fig. 5

Antibacterial mechanism of Te NNs. ITC spectra and fitting curve of Te NNs to (a) PG and (b) LPS. (c) Fluorescence images and (d) luminescence intensity of ROS in Te NNs group, S. aureus control group, S. aureus+Te NNs group, E. coli control group and E. coli+Te NNs group. (e) Bacterial agar gel electrophoresis profiles of genomic DNA obtained from direct DNA extracts of the samples; allelic bands were: Lane Marker, 15 ​000 bp DNA ladder; Lane 1, S. aureus control group; Lane 2, S. aureus+Te NNs group; Lane 3, E. coli control group; Lane 4, E. coli+Te NNs group.

In addition, the antibacterial activities of Te nanoparticles are possibly related to the formation of ROS [35,[52], [53], [54]]. A high ROS level can increase oxidative stress in cells, which can damage both the cell membranes and the proteins, then cause bacterial death. Fluorescence images of ROS in Fig. 5c show green fluorescence in both S. aureus and E. coli with the treatment of Te NNs. Moreover, the E.coli+Te NNs group emits stronger green fluorescence, indicating more ROS generation, hence enhancing the sterilization ability. Nevertheless, the Te NNs per se emit negligible fluorescence of ROS, whose luminescence intensity is far lower than S.aureus+Te NNs and E.coli+Te NNs groups (Fig. 5c and d). Furthermore, after the introduction of Te NNs, the genomic levels of S. aureus and E. coli are all weaker than that of respective control group, especially for E.coli+Te NNs group. In general, the Te NNs cover and adhere to the surfaces of S. aureus and E. coli and further accelerate ROS generation of bacteria to cause membrane and DNA damage, finally eliminating the bacteria. Moreover, the better morphological damage and biofilms destruction ability toward E. coli may be owing to more ROS generation. And as shown in the green fluorescence in Figure S6c, the intracellular ROS of S. aurues stimulated by Te NNs is more than that stimulated by Te–SO NNs, contributing to better antibacterial ability to S. aureus of Te NNs. Compared to current antibacterial agents which promote ROS generation to eliminate bacteria under external stimuli (e.g. photo, sound, microwave) [[55], [56], [57], [58], [59]], the Te NNs perform more flexibility in the application.

We further investigate the SEM images (Figure S7) which shows the puncturing behavior of Te NNs. When the bacteria co-cultured with Te NNs, the nanoneedles have a contact with the bacterial cells and as shown in the partial enlarged views of Figure S7, the needle-like structure punctures bacterial membranes, visibly deforming the cells, especially the E. coli cells. Therefore, the antibacterial mechanism of Te NNs attributes to the chemical (ROS) damage as well as physical damage on bacteria.

Healing therapy for infected wounds

We establish S. aureus infected wound model by full-thickness skin wounds excision and immediate S. aureus infection. To confirm the in vivo antibacterial and wound healing applicability, the Te NNs/PVA hydrogel is prepared and placed on the wounds 24 ​h post-surgery. Fig. 6a records representative photographs of the wounds at different time points. The trauma in the control and PVA hydrogel groups presents a large area of unhealed skin defect with severe infection after 4 days of treatment, while the trauma in the Te NNs/PVA hydrogel group heals faster at each time point. To further confirm the sterilization ability of Te NNs/PVA hydrogel, the residual bacteria are collected from the wound and cultured in the NB agar plates. Densely packed colonies are observed in the control and PVA hydrogel groups even at the treatment of day 14 (Fig. 6b), which cause continuous exudate from the infected wounds, prolonging the healing process. Obviously, at each time point, the number of residual bacteria at the wounds treated with Te NNs/PVA hydrogel was apparently lower than the others (Fig. 6b,d). Especially after 14 days of treatment, the inflammation of wounds is eliminated, attributing to a better recovery from the bacterial infection with a healing ratio of 98% (Fig. 6a,c).

Fig. 6

Therapeutic effect of Te NNs/PVA hydrogel on S. aureus infected wounds in vivo. (a) Representative photographs of wounds treated with PBS, PVA hydrogel and Te NNs/PVA hydrogel at days 0, 4, 7, 10, and 14; scale bar, 1 ​cm. (b) Photographs of bacterial cultures taken from the skin tissue with different treatments during the same time period. (c) The wound healing ratio of rats from day 4 to day 14. (d) The number of residual bacteria at the wound sites of different treatment groups. Data are represented as mean ​± ​standard deviation (n ​= ​5 in each group); ∗p ​< ​0.05, ∗∗p ​< ​0.01 compared to the Te NNs/PVA group.

The H&E staining and Masson's trichrome staining of the infected wound tissue are performed to observe the re-epithelization, fibroblast immigration, connective tissue synthesis, infiltration of inflammatory cells and collagen production. After 7 days of treatment, the obvious neutrophil infiltration surrounding the wounds can be observed in the control and PVA hydrogel groups (Fig. 7a), which indicates a severe bacterial infection. In comparison, the Te NNs/PVA hydrogel group exhibits negligible neutrophils and more fibroblasts, thus confirming the wound healing effects of Te NNs. On the 14th day, except for the control group, the other two groups form a layer of complete epithelium. Moreover, the Te NNs/PVA hydrogel group exhibits the maximum amount of mature blood vessels, hair follicles and well-proliferated fibroblasts, demonstrating the best wound healing effect. The formation and distribution of collagen on wounds are evaluated using Masson's trichrome staining in Fig. 7b. The shade of blue in the Te NNs/PVA hydrogel group is apparently darker than the other two groups after both 7 days and 14 days of treatment, revealing Te NNs/PVA can improve collagen formation to accelerate the wound healing process.

Fig. 7

The histological images of the skin tissue on S. aureus infected wounds. (a) H&E stained images and (b) Masson's staining images showing the degree of S. aureus infection of skin tissue after treating with different dressings for 7 and 14 days. Scale bar, 100 ​μm. Yellow arrows: neutrophils; Blue arrows: fibroblasts.

Furthermore, S. aureus and E. coli co-infected wound model is also established to assess the antibacterial ability of Te NNs in vivo since mixed bacterial infection is more likely to cause severe consequences, hindering wound healing [60,61]. Fig. 8a shows a large area of skin defect with inflammation, suppuration, and ulceration in the control group during the first week post-surgery. As for Te NNs/PVA hydrogel group, the trauma becomes dry and scab at day 4 and is healed completely after 14 days of treatment, whereas the control and PVA hydrogel groups do not heal well with a remaining area (Fig. 8a,c). The bacteria residues in wounds further confirm the bacterial elimination ability of Te NNs as the negligible colonies can be observed in the Te NNs/PVA hydrogel group on the tenth day (Fig. 8b,d). However, bacterial infection still exists in the control and PVA groups even after 14 days of treatment.

Fig. 8

Therapeutic effect of Te NNs/PVA hydrogel on S. aureus & E. coli co-infected wounds in vivo. (a) Representative photographs of wounds treated with PBS, PVA hydrogel and Te NNs/PVA hydrogel at days 0, 4, 7, 10, and 14; scale bar, 1 ​cm. (b) Photographs of bacterial cultures taken from the skin tissue with different treatments during the same time period. (c) The wound healing ratio of rats from day 4 to day 14. (d) The number of residual bacteria at the wound sites of different treatment groups. Data are represented as mean ​± ​standard deviation (n ​= ​5 in each group); ∗p ​< ​0.05, ∗∗p ​< ​0.01 compared to the Te NNs/PVA group.

The wound skin tissues of the three group mice are obtained and stained by H&E on days 7 and 14 (Fig. 9a). In the control and PVA groups, a lot of white blood cells are produced at the 7 days of infection and they mainly existed as neutrophils, demonstrating the inflammatory response by S. aureus and E. coli. And the congestion appears notably in the control group due to serious inflammation. In the following days, the fibroblasts increase with the decreasing of neutrophils and the squamous epithelium forms during the healing process. In the Te NNs/PVA hydrogel group, the foci almost disappear and the neonatal tissues are smooth at day 14. Simultaneously, a large number of intact capillaries filled with red blood cells appear, proving the excellent treatment effect of Te NNs/PVA hydrogel. The blue staining in Fig. 9b shows that Te NNs/PVA hydrogel group performs a higher density of collagen compared to the control and PVA hydrogel groups at days 7 and 14. Moreover, after 14 days of treatment, the newly formed collagen fibers of the wounds in Te NNs/PVA hydrogel group have a perfect fusion with normal tissues, indicating that Te NNs/PVA hydrogel is conductive to collagen formation, thus promoting wound healing.

Fig. 9

The histological images of the skin tissue on S. aureus & E. coli co-infected wounds. (a) H&E stained images and (b) Masson's staining images. The results show the degree of S. aureus & E. coli co-infection of skin tissue after treating with different dressings for 7 and 14 days. Scale bar, 100 ​μm. Yellow arrows: neutrophils; Blue arrows: fibroblasts.

In addition, the H&E staining images of the main organs including heart, liver, spleen, lung, and kidney in the control and treated groups are depicted in Fig. 10. In both the S. aureus infected and S. aureus & E. coli co-infected model, Te NNs/PVA hydrogel has no hazards to major organs since the H&E sections of the heart, liver, spleen, lung, and kidney in the treatment group show no obvious histological changes compared to control group. Therefore, Te NNs/PVA hydrogel provides a safe and effective strategy for the treatment of infected wounds.

Fig. 10

H&E staining of the heart, liver, spleen, lung, kidney tissue slices of different treatment groups after 14 days. Scale bar, 100 ​μm.

Conclusions

In conclusion, a kind of biocompatible Te NNs is synthesized by a facile method for long-term stable antibacterial application. The as-prepared Te NNs are inherently inert to ROS generation and also do not stimulate the generation of ROS in mammalian cells, which exhibits low cytotoxicity. It is found that the biocompatible Te NNs can adhere to the surface of S. aureus and E. coli and subsequently promote the generation of ROS in bacteria and the needle-like structure can cause the physical damage of bacterial membranes, which results in excellent antibacterial ability. Moreover, this bacteria-killing activity is still maintained at a high level with the surface oxidation of Te NNs in PBS even for 6 months. Furthermore, in vivo wound therapeutic results demonstrate that the Te NNs/PVA hydrogel can eliminate bacteria as well as promote wound healing. We believe this work helps us understand the antibacterial behaviors of Te NNs and deliver effective therapeutic guidance for application in infectious diseases.

Credit author statement

Lihua Li: Supervision and guidance. Zhijun Ma: Supervision and guidance. Ling Huang: Materials preparation, characterization analysis, biological examination, animal related experiments, data curation, writing – original draft. Meng Liu: Materials preparation, characterization analysis, biological examination, writing-supplement. Zhibin Feng: Animal related experiments. Xingyi Xu: Characterization analysis. Lingling Chen: Animal related experiments.

Declaration of competing interest

The authors declare that they have no conflict of interest.