Biomaterials

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PAPER

Potent intrinsic bactericidal activity of novel

Cite this: Biomater. Sci., 2023, 11,
copper telluride nano-grape clusters with facile
1828 preparation†
Yanwen Zhou,‡a,b Haozhuo Lei,‡b Meng Wang,b Yanbin Shi*b and
Zhaohui Wang *a,b

Bactericidal nanomedicines often suffer from a complicated design and insufficient intrinsic inhibitory
efficacy. Herein, novel anti-bacterial copper telluride (CuTe) nano-clusters are reported, featuring superior
bactericidal efficiency, facile preparation, and unique mechanism. These nanoparticles, well dispersable in
water, resembled grape clusters with rough surfaces. The CuTe nano-grape clusters exhibited ultra-high
sterilization efficacy at ultra-low concentration, particularly for Gram-negative bacteria, and were more
potent than conventional anti-microbial nanoparticles. Also, the grape clusters effectively inhibited the
bacterial biofilm development. Further investigation revealed the synergized mechanisms of reactive
oxygen species (ROS) generation and glutathione (GSH) depletion. Interestingly, electron microscopy
revealed that the grape clusters served as bacterial hunters by tightly adhering to bacterial surfaces. The
bacteria subsequently suffered from the leakage of various intracellular components including nucleic
Received 5th October 2022, acid, proteins, and potassium. Most encouragingly, CuTe drastically reduced bacterial number in a mouse
Accepted 31st December 2022
model with lethal intraperitoneal infection and increased the mouse survival rate to 90%. This finding
DOI: 10.1039/d2bm01617f could inspire the development of highly potent bactericidal inorganic formulations with simplified struc-
[Link]/biomaterials-science ture, multiple antibacterial mechanisms, and promising application potential.

1. Introduction mize their efficacy. For example, the synergy of glutathione

(GSH) depletion, nitric oxide (NO) generation, and photo-
Bacterial infection as a global health problem is drawing dynamic therapy (PDT) was realized in a nano-system and
increasing attention. Until now, antibiotics have served as the effectively eradicated the biofilm of drug resistant bacteria.6
main therapeutics against various bacteria, although their Bacterial membrane coating was employed to increase the
overuse has caused the emergence of multi-drug resistance in uptake of rifampicin-loaded nanoparticles by Gram-negative
pathogenic microorganisms.1,2 To tackle this problem, unre- bacteria.7 On the other hand, nano-formulations with intrinsic
mitting efforts have been made to discover new agents to anti-bacterial efficacy are equally important. Silver formu-
enrich the arsenal against bacteria. Among these agents, nano- lations are the most widely reported inorganic materials to
materials are particularly attractive compared to their small- intrinsically kill bacteria.8,9 However, there are major limit-
molecule counterparts, with unique advantages of multiple ations for silver, such as safety concerns related to excessive
action modes3,4 and rare resistance.5 In general, in anti-bac- release of toxic Ag+ ions10,11 and relatively insufficient anti-bac-
terial applications, nano-constructs serve as either drug car- terial efficiency.12 Recently, catalytic inorganic particles have
riers or intrinsic bactericidal material. Nano-carriers can syner- emerged as new anti-bacterial materials, which possess pro-
gistically combine different therapeutic modalities to maxi- perties like enzymes including peroxidase, oxidase, and gluta-
thione oxidase.13,14 The nano-enzymes or nanoparticles with
catalytic activity can catalyse the conversion of substrates into
a
Key Laboratory of Biomedical Engineering of Hainan Province, School of Biomedical reactive oxygen species (ROS), the major toxic elements for
Engineering, Hainan University, Haikou, 570228, China. sterilization.15,16 However, efficient ROS generation heavily
E-mail: wangzhaohui@[Link] relies on the proximity between nano-materials and bacteria
b
School of Pharmacy, Lanzhou University, Lanzhou, 730000, China. and is limited by insufficient ROS lifespan and diffusion
E-mail: shiyb@[Link]
distance.17,18 Therefore, higher material doses, exogenous sub-
† Electronic supplementary information (ESI) available. See DOI: [Link]
10.1039/d2bm01617f strates such as H2O2, and the aid of photo-therapy are often
‡ Equal contributions. needed to further enhance the anti-bacterial efficacy of the

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Biomaterials Science Paper

nano-enzymes,19,20 which introduces multiple components. A inhibition has been barely explored, to the best of our knowl-
few recent studies have improved the bacterial binding and edge. In addition, GSH depletion has been proved effective to
ROS generation ability of nano-constructs by the morphology enhance the photodynamic inhibition of bacteria biofilm.6 In
design and defect engineering of nano-enzymes.21,22 this study, by a facile method at room temperature with simple
Nonetheless, the anti-bacterial efficacy was still unsatisfactory protocols, we prepared aqueous CuTe nanoparticles, which
without the help of H2O2 or laser irradiation. have a unique “grape cluster” morphology and can be stably
To date, nano-materials exhibiting strong intrinsic steriliza- dispersed in water and conveniently used for infection therapy
tion efficacy (minimum inhibitory concentration lower than (Scheme 1). Most importantly, the CuTe nano-grape clusters
10 μg mL−1) without external synergy are scarcely reported8,14 serve as excellent bacteria hunters, exhibiting superior anti-
and the mechanism investigation has been insufficient. It is bacterial efficacy without the aid of external agents. Different
thus urgently needed to exploit more of such advantageous from normal spherical nanoparticles, our CuTe has a strong
materials for bacterial killing and explore the underlying anti- ability to capture bacteria, largely owing to its specific struc-
bacterial mechanism that remains obscure. ture with rough surfaces,21,27 as seen by electron microscopy.
In addition, many nanoparticles for biomedical appli- Particularly, a microplate reader accurately recorded its high
cations are prepared in non-polar solvents at ultra-high inhibition rates against E. coli and Acinetobacter baumannii at
temperature.23,24 This leads to laborious modifications to an extremely low concentration of 0.625 μg mL−1. Agar plate
increase solubility in biological fluids, such as micelle encap- observations corroborated the microplate data and highlighted
sulation or ligand exchange for phase transferring. These pro- the obvious superiority of CuTe compared to the control anti-
cesses require complicated expertise and add unwanted com- biotic chloramphenicol. We investigated the mechanism of the
ponents. The synthesis of aqueous semiconductor nanocrys- grape clusters’ action with respect to enzyme-mimicking pro-
tals with simple modification, excellent solubility, and perties, physical association with bacteria, and physiological
ambient preparation condition is still difficult.25 changes in bacteria. Finally, the in vivo anti-bacterial efficacy
Copper telluride nanoparticles have recently been reported was evaluated in an intraperitoneal infection model.
for anti-tumour therapy with combined catalytic activity and
photothermal efficacy,23,26 in which the copper telluride could
catalyse the generation of ROS and deplete GSH. However, the 2. Experimental section
relatively laborious synthesis or modification for water solubil-
ization may limit its broad biological use, especially an anti- 2.1. Materials
bacterial application that requires direct interaction between Tellurium powder (99.99% metals basis), copper chloride
the material and bacteria. On the other hand, copper telluride (98%), chloramphenicol (98%), and o-phenylenediamine
prepared by the aqueous precipitation method for bacterial (OPD, 99.5%) were purchased from Aladdin.

Scheme 1 Schematic illustration of easy preparation and application of CuTe.

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Polyvinylpyrrolidone (PVP, average molecular weight ∼55 000) resents the absorbance of the control group at 600 nm, Ab
was purchased from Aldrich-Chemistry, Merck. Sodium boro- treated is the absorbance of bacteria treated with different
hydride (NaBH4, AR) was purchased from Damao Chemical agents, Ab agent is the absorbance of the agent with the
Reagent Factory. Acinetobacter baumannii (ATCC19606), corresponding concentration of a certain group and Ab blank
Staphylococcus aureus subsp. aureus (ATCC29213) and is the absorbance of the NB culture used in the experiment.
Escherichia coli (ATCC25922) were purchased from Beina
Chuanglian Biotechnology (Beijing) Institute. Reduced gluta- 2.4. Anti-bacterial assay in agar plate
thione (GSH) assay kit and 2,7-dichlorodihydrofluorescein dia- E. coli (107 order of magnitude CFU per mL), Acinetobacter bau-
cetate (DCFH-DA, ≥97.0%) were purchased from Solarbio Life mannii (107 order of magnitude CFU per mL), and S. aureus
Sciences. Titanium dioxide (20–40 nm, 99.8% metals basis) (108 order of magnitude CFU per mL) were incubated with
and gold nanoparticles (5 nm) were purchased from Shanghai different concentrations of agents in NB medium. After
Acmec Biochemical. SYTO-9 was purchased from Thermo 2–3 hours of incubation at 37 °C, a 1000-times dilution was
Fisher Scientific. Silver nanoparticles (30 nm) were purchased conducted for the bacterial solution in each group. Then, the
from Acmec. BALB/c mice (female, 18–22 g) were purchased diluted bacteria were spread evenly on NA agar plates and
from Lanzhou Veterinary Institute, Chinese Academy of incubated at 37 °C for 10–12 hours until the colonies were
Agricultural Sciences. easily observable, followed by imaging by mobile phone.

2.2. Preparation of CuTe nano-grape clusters 2.5. ROS generation and GSH depletion assay
Tellurium powder (63.8 mg) was added to a three-neck flask 8.5 mL of CuTe solution (20.5 μg mL−1, Cu and Te only) was
containing 36 mL ethanol and 12 mL ultrapure water. The added to 1.5 mL OPD solution (1 mg mL−1). Then, different
system was subjected to N2 flow for at least 30 minutes. Then, amounts of 30% H2O2 solution were added to the above
56.75 mg NaBH4 was dissolved in 2 mL ultrapure water and mixture, which was quickly subjected to UV-VIS spectropho-
injected into the flask while stirring and the reaction contin- tometry, where the absorbance at 418 nm was recorded along
ued for 5–6 hours until the solution became red. 134.45 mg with the reaction time. The control solution was a mixture of
CuCl2 and 40 mg PVP were mixed in 2 mL water, injected into CuTe and OPD. Different time-absorbance curves were
the flask, and reacted for 1.5 hours. After the reaction, the obtained using different H2O2 amounts. The Michaelis–
crude product was centrifuged at 5500 rpm for 15 minutes and Menten equation was obtained by a non-linear fitting of the
the precipitates were discarded. The supernatants were col- speed of absorbance changes with H2O2 concentrations. For
lected, again followed by 5500 rpm centrifugation for the GSH depletion assay, the absorbance curves with a 412 nm
15 minutes after which the supernatants were again collected. characteristic peak were recorded with different CuTe concen-
The final product was collected after filter paper purification trations after a 10-minute reaction with GSH, using a commer-
and dialysis using a 100 000 D dialysis bag for at least cial GSH quantification kit.
48 hours. The concentration was determined by inductively
coupled plasma-optical emission spectroscopy (ICP-OES). The 2.6. ROS imaging
particle size was determined by a particle size analyzer 1 mL E. coli (109 order of magnitude CFU per mL) and
(Brookhaven, USA) and the zeta potential was measured by a S. aureus (1010 order of magnitude CFU per mL) were incu-
Zetasizer Nano ZS (Malvern, England). bated with 1 mL CuTe with a final concentration of 50 μg ml−1.
Then, 40 μL DCFH-DA (1 mM) was added into the solution
2.3. Determination of inhibition rates in microplate and incubated at 37 °C for 30 minutes in the dark, followed by
E. coli (107 order of magnitude CFU per mL), Acinetobacter bau- fluorescence microscopy observation.
mannii (107 order of magnitude CFU per mL), and S. aureus
(108 order of magnitude CFU per mL) were cultured in a 2.7. Biofilm formation assay with crystal violet staining
96-well microplate and incubated in NB culture with agents of 100 μL E. coli (108 order of magnitude CFU per mL) were
different concentrations. For E. coli and Acinetobacter bauman- seeded in a 96-well plate. The control group was added with
nii, the concentration gradient of CuTe was selected to be 20, 100 μL medium. Treatment groups were incubated with CuTe
10, 5, 2.5, 1.25, and 0.625 μg mL−1 with 3 parallel wells for or chloramphenicol with final concentrations of 40, 20, 10, 5,
each concentration. For S. aureus, CuTe concentrations of 80, 2.5, and 1.25 μg mL−1. After 54 h incubation, the culture
40, 20, 10, 5, and 2.5 μg mL−1 were used. The CuTe concen- medium was removed and the wells were washed. Then, after
trations used in this study were calculated by adding the Cu drying, 125 μL of crystal violet was added into each well for
and Te concentrations obtained by ICP-OES and PVP content 15 minutes, followed by 30% glacial acetic acid dissolution.
was not added. The microplate was placed in the incubator at The solution was removed to a new 96-well plate and the
37 °C for 9 hours. Afterwards, the 600 nm absorbance of bac- 600 nm absorbance was recorded.
teria in each group was determined by microplate reader. The
inhibition rate was calculated by the following formula: inhi- 2.8. Anti-biofilm assay with fluorescence imaging
bition rate (%) = (Ab control − Ab treated + Ab agent − Ab E. coli (108 order of magnitude CFU per mL) were seeded into
blank)/(Ab control − Ab blank). In this formula, Ab control rep- a 96-well microplate in 200 μL NB medium and incubated at

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37 °C for at least 48 hours to form a biofilm. During incu- after 8000 rpm centrifugation, the supernatants were collected
bation, 50 μL of fresh medium was supplemented to the wells for ICP-OES quantification after dilution.
with bacteria after 24 hours. After the 48-hour incubation, the
free bacteria remaining in the medium were removed by 2.12. Nucleic acid and protein leakage assay
washing with PBS solution twice. Wells with a formed biofilm
E. coli (109 order of magnitude CFU per mL) and S. aureus
were treated with CuTe (200 μg mL−1) or an equal volume of
(1010 order of magnitude CFU per mL) were subjected to 50 μg
PBS. After a 12-hour incubation, 30 μL SYTO-9 (50 μM) and
mL−1 CuTe for 2, 30, 60, and 120 minutes. At each time point,
20 μL PI (50 μM) were added and incubated at 37 °C for
the bacteria of the corresponding group were centrifuged at
40–50 minutes. Then, the biofilm was observed under fluo-
9000 rpm for 10 minutes. Then, the supernatants were col-
rescent microscope.
lected and the concentrations of nucleic acid and protein were
determined by a Nanodrop 2000 instrument. Bacteria in PBS
2.9. SEM and TEM imaging of bacteria
without treatment served as control.
Bacteria were washed and resuspended in 1.5 mL PBS solution.
Then, 1.5 mL CuTe solution was added to the bacteria, to final 2.13. Bacterial capture assay
concentrations of 84.5 μg mL−1 for S. aureus and 42.3 μg mL−1
for E. coli. The bacteria were then subjected to 37 °C incu- 100 μL E. coli (107 order of magnitude CFU per mL) or
bation with 180 rpm shaking for 10–12 hours. Then, the bac- S. aureus (107 order of magnitude CFU per mL) were added
terial samples were washed twice with PBS solution and fixed into 1 mL of CuTe solutions of different concentrations (50,
with glutaraldehyde (2.5%). Three parallel samples for E. coli 25, and 12.5 μg mL−1). An equal amount of bacteria added
and S. aureus were collected. The fixed samples were subjected into culture medium served as the control. After 1 minute of
to standard preparations for SEM and TEM imaging. For TEM, mixing, the mixtures were kept still for 9 minutes. Then, the
sample preparation, agarose pre-embedding, post fixing with upper section of the bacterial solution was collected and
osmic acid, dehydration and embedding were accomplished, diluted for spreading on NA agar plates. The colony number of
followed by model polymerization, sectioning, staining, and the control group was recorded as N0 and the number after
imaging. The SEM sample preparation involved osmic acid CuTe treatment was N1. The capture rate was calculated as 1 −
post fixing, dehydration, drying, metal coating, and imaging N1/N0 × 100%.
collection and analysis.28
2.14. In vivo anti-bacterial assay
2.10. Biocompatibility assay 15 BALB/c mice were randomly divided into control, CuTe, and
Animal experiments followed the Animal Management Rules chloramphenicol groups, respectively. Then, the mice in each
of the Ministry of Health of People’s Republic of China and group were intraperitoneally injected with 200 μL E. coli (107
the requirements of the ethics committee of the School of order of magnitude CFU per mL) in NB medium. One hour
Pharmacy, Lanzhou University. For in vivo acute toxicity evalu- after bacterial injection, the control group was intraperitone-
ation, BALB/c mice were intraperitoneally treated with 500 μL ally injected with 500 μL saline, the CuTe group was intraperi-
CuTe solution of 100 μg mL−1. The healthy mouse was used as toneally injected with 500 μL CuTe solution of 100 μg mL−1,
a control. Five days after treatment, the mice were sacrificed and the chloramphenicol group was injected with 500 μL
and major organs, including liver, spleen, lung, and heart, chloramphenicol solution of 100 μg mL−1. 24 hours after treat-
were collected followed by hematoxylin–eosin (H&E) staining. ment, the mice were sacrificed, and the peritoneal fluid was
For the hemolysis assay, rabbit erythrocytes (6%) were incu- obtained by opening the abdomen and washing with 4 mL
bated with CuTe with final CuTe concentrations of 50, 25, 12.5, PBS solution. The collected fluid was stored for further exam-
6.25, 3.125, and 1.5625 μg mL−1; equal volumes of Triton ination. The major organs, including liver, spleen, and kidney,
X-100 and PBS were added to serve as positive and negative were obtained, washed, and homogenized in 5 mL PBS solu-
controls, respectively. Afterwards, the plate was incubated at tion. The peritoneal fluid and homogenates of the major
37 °C for 1 hour and subjected to centrifugation. The super- organs were 10-times diluted, spread onto agar plates and
natants were collected and the absorbance at 540 nm was incubated for colony number counting.
recorded for the hemolysis ratio calculation.
2.15. Survival assay
2.11. Extracellular K+ assay 20 BALB/c mice were randomly divided into CuTe and control
E. coli (108 order of magnitude CFU per mL) and S. aureus (109 groups. Mice in each group were intraperitoneally injected
order of magnitude CFU per mL) in NB culture were centri- with 200 μL E. coli (108 order of magnitude CFU per mL),
fuged, washed with saline, and resuspended in equal volumes which was a lethal dose. 20 minutes after bacterial injection,
of saline. Then, 3 mL of bacterial solution in saline were mice in the control group were injected with 500 μL saline and
added with 3 mL saline or 3 mL CuTe solution for a final CuTe mice in the CuTe group were intraperitoneally injected with
concentration of 100 ug mL−1. The two groups were subjected 500 μL CuTe solution of 100 μg mL−1. Mouse survival was
to 37 °C incubation for 2 hours with 120 rpm shaking. Then, observed for 14 days.

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3. Results and discussion (SEM) at different magnifications revealed the rough surfaces
of these clusters (Fig. 1b), which may favour bacterial binding.
3.1. Preparation and characterization of CuTe nano-grape X-ray photoelectron spectra (XPS) suggested that the valence
clusters state of Cu was +2, as validated by the 2p 1/2 and 3/2 binding
The ambient synthesis of CuTe was easily achieved and the as- energies together with the strong satellite peaks (Fig. 1e). Also,
prepared CuTe nano-grape clusters were well dispersed in the UV-vis spectrum is shown in Fig. S2.† It is worth noting
water, exhibiting a blackish green colour (insert in Fig. 1a). that the preparation of the CuTe clusters occurred at room
Transmission electron microscopy (TEM) demonstrated the temperature by a one-pot strategy.
grape-cluster shaped nano-structures of CuTe (Fig. 1a and
Fig. S1a, b†) with mean diameter of about 263.21 nm and poly- 3.2 Anti-bacterial and anti-biofilm efficacy
dispersity around 0.1633 (Fig. 1d). The zeta potential was Although copper telluride nanomaterials have been
measured to be 7.61 mV. High-resolution TEM (HRTEM) reported,29–31 studies on their biomedical application were
found lattice fringes of single nano-particles of the grape-clus- scarce,23 especially for anti-bacterial purposes. We demon-
ters with interplanar distances of 0.24 nm (Fig. 1a), but X-ray strate that the CuTe nano-grape clusters exhibited potent bac-
powder diffraction (XRD) only provided 1 diffraction peak tericidal efficacy against E. coli and Acinetobacter baumannii. At
(Fig. S1c†), indicating that most parts of the nanostructures a very low concentration of 0.625 μg mL−1, the inhibition rates
were amorphous. The composition of CuTe was further vali- of E. coli and Acinetobacter baumannii reached 93.6333% and
dated by dark-field TEM and elemental mapping (Fig. 1c). It 88.0167%, respectively. For Acinetobacter baumannii, a
was observed that Cu (red), Te (yellow), and O (green) elements 99.3967% inhibition rate was achieved at 1.25 μg mL−1 (Fig. 2a
were homogeneously distributed in the grape-clusters and and e). In contrast, the conventional nanoparticles with known
were well merged. Cu and Te signals confirmed the formation bacterial inhibition effect, such as TiO2, gold, and silver nano-
of CuTe nanoparticles and the O signal indicated polyvinylpyr- particles, barely suppressed the bacteria in this study at such
rolidone (PVP) modification. Scanning electron microscopy low concentrations (Fig. 2b, c, f and g). Statistical analysis con-

Fig. 1 Characterization of CuTe nano-grape clusters. (a) TEM image, CuTe solution appearance, and high-resolution TEM image. (b) SEM images of
CuTe at different magnifications. (c) Dark-field image and elemental mapping. (d) Size distribution and polydispersity. (e) XPS spectra of Cu 2p and
Te 3d.

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Fig. 2 (a–c) Inhibition effects of CuTe, gold nanoparticles and TiO2 on E. coli. (d) Comparison of the inhibitory effects of CuTe, chloramphenicol,
TiO2, gold nanoparticles, and silver nanoparticles on E. coli. (e–g) Inhibition effects of CuTe, gold nanoparticles, and TiO2 on Acinetobacter bauman-
nii. (h) Comparison of the inhibitory effects of CuTe, TiO2, gold nanoparticles, and silver nanoparticles on Acinetobacter baumannii. (****P < 0.0001).
(i–k) Agar plate images of the bactericidal effects of different agents.

firmed the significantly stronger bactericidal efficacy of CuTe CuTe, compared with the dense bacterial distribution in the
compared with other nanoparticles, even the antibiotic chlor- control group; when the concentration increased, the plate
amphenicol (Fig. 2d and h). CuTe also exhibited effective steri- became clearer in sharper contrast to the control (Fig. 2i).
lization against S. aureus, the Gram-positive model bacteria, Similar inhibition efficacy was observed for Acinetobacter bau-
although the efficacy was lower than for Gram-negative bac- mannii (Fig. 2k). CuTe was obviously more potent than the
teria (Fig. S3†). In the spread plate experiment, only a small positive control drug chloramphenicol, as many E. coli colo-
number of E. coli colonies were observed with 1.25 μg mL−1 nies still appeared on the control plate even at a high chloram-

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phenicol concentration of 20 μg mL−1 (Fig. 2j). Gram-negative depletion. OPD, the oxidation product of which can be
bacteria are characterized by a poorly-permeable cell mem- detected at 418 nm absorption, was used to indicate the gene-
brane compared with positive bacteria, which impedes the ration of ROS.37 The time-absorbance data in Fig. 4a revealed
action of many antibiotics that are effective in Gram-positive that at fixed CuTe concentration, with the increase of H2O2
germs.32 Therefore, the ultra-strong inhibitory effect of CuTe volume, the 418 nm absorbance increased more quickly. As
against Gram-negative bacteria provides an alternative agent to the 418 nm absorbance can indirectly reflect H2O2 consump-
circumvent these hard-to-treat bacteria. Also, in future appli- tion, the non-linear Michaelis–Menten equation was fitted as
cations, CuTe may serve as a drug carrier for delivering various in Fig. 4b. The Km and Vmax values were also calculated from
antibiotics across the barrier of Gram-negative bacteria. Anti- the equation (Fig. 4b). GSH depletion efficacy was evaluated by
biofilm evaluation is an important aspect to evaluate a new a reduced glutathione (GSH) assay kit. The GSH characteristic
antibacterial agent because conventional antibiotics often fail peak decreased with elevated CuTe concentration (Fig. 4c),
to destroy the barriers established when biofilm forms.24,33–35 proving the GSH exhaustion ability of CuTe. These two features
For an already established biofilm, CuTe could effectively of CuTe, ROS generation and GSH depletion, could be perfectly
break this dense defence, as only marginal SYTO-9 fluo- synergized to maximize the killing of bacteria by ROS. This is
rescence, the signal of which indicates the distribution of because reductive GSH can greatly attenuate ROS and protect
living bacteria, could be observed. In contrast, the bright green cells from damage.38,39 Particularly, E. coli biofilm is character-
fluorescence of SYTO-9 was widely distributed in the control ized by high GSH concentration.6 Therefore, from the perspec-
group with little PI fluorescence. Accordingly, the PI signal was tive of chemical catalysation, the outstanding anti-bacterial
stronger in the CuTe group than in the control (Fig. 3a). In and anti-biofilm performance of CuTe could be explained. In
addition, crystal violet quantification was used to evaluate the addition, fluorescence imaging with an ROS probe showed
inhibition of biofilm formation, in which CuTe was added to obvious signals in both E. coli (Fig. 4d) and S. aureus (Fig. 4e)
bacteria before the biofilm formed. The results showed that cells after CuTe treatment. Interestingly, we found that in the
CuTe significantly reduces biofilm mass, possibly because a confocal dish, CuTe caused obvious aggregation of bacteria
certain number of bacteria were killed during biofilm for- (Fig. 4f ), like hunters collecting prey. This phenomenon is
mation. At low concentration, the biofilm inhibition rate of most likely due to the binding and capturing ability of CuTe.
CuTe was significantly higher than that of the positive drug The bacterial hunting effects were further quantitatively con-
chloramphenicol (Fig. 3b). These results validated the anti- firmed in Fig. 4g (E. coli) and 4h (S. aureus), confirming the
biofilm ability of CuTe nano-grape clusters. bacteria-capturing observation. The bacterial hunting ability of
CuTe is most likely endowed by its rough surface and positive
3.3 Mechanism exploration for CuTe nano-grape clusters zeta potential. To evaluate the subsequent physiological
Next, we sought to investigate the underlying mechanism of changes in bacteria after capture, the leakages of intracellular
the potent anti-bacterial performance of CuTe. The catalytic or components, including nucleic acid, protein, and K+, were
enzyme-like activities of inorganic nano-materials have been determined. With CuTe incubation, the extracellular concen-
reported in anti-tumor and anti-bacterial studies.14,36 Here, trations of nucleic acid (Fig. 4i) and protein (Fig. 4j) obviously
CuTe was tested for the promotion of ROS formation and GSH increased from 2 to 120 minutes in E. coli, compared to

Fig. 3 Biofilm inhibition examined by (a) fluorescence imaging and (b) crystal violet staining (**P < 0.01).

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Fig. 4 (a) Absorbance changes of ROS indicator with time under different H2O2 amounts. (b) Michaelis–Menten fitting of absorbance change vel-
ocity with H2O2 concentration. (c) Evaluation of GSH depletion using assay kit with characteristic absorbance spectra. (d) and (e) ROS generation in
bacteria indicated by fluorescence signal. (f ) Direct visualization of bacteria capture. (g) Quantification of E. coli capture rate. (h) Quantification of
S. aureus capture rate. (i) and ( j) Leakage of nucleic acid and protein from E. coli. (k) and (l) Leakage of nucleic acid and protein from S. aureus.

minimal changes in the control group. Similarly, in S. aureus, SEM images of E. coli in the control group (Fig. 5a), the CuTe
evident release of nucleic acid (Fig. 4k) and protein (Fig. 4l) treatment group bacteria retained the characteristic “rod”
was also observed. The leakage of K+ can indicate the loss of shape, but became aggregated and the overall bacterial struc-
cytoplasm in bacteria.8,40 Interestingly, after CuTe treatment, ture shrank (Fig. 5b). Also, CuTe clusters were observed on the
we found obvious K+ leakage in S. aureus but not in E. coli bacterial surfaces as “bacterial hunters”, which probably
(Fig. S4†). Taken together, these data suggest the mechanism served to capture bacteria and decrease their activity. The dis-
of CuTe nano-grape clusters is strongly associated with ROS tribution of CuTe was further validated by EDS mapping. CuTe
generation, GSH depletion, and the loss of intracellular treatment led to bright Cu and Te signals (EDS column in
components. Fig. 5b) that were not detected in the control group. Next, the
changes in the inner structures of E. coli were visualized by
3.4. Visualization of bacterial hunting by CuTe TEM (TEM column in Fig. 5). Compared to the control group,
Bacterial structures, including flagella and cilia, make them bacteria with CuTe treatment showed evidently decreased
favour rough surfaces compared with human cells.17 The intracellular electron density, indicating possible excess water
rough CuTe surfaces inspired us to observe the physical associ- accumulation and local cytoplasm dissolution (Fig. 5b). Also,
ation of CuTe grape clusters with bacteria. Compared with plasmolysis could be observed in the CuTe group compared to

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Fig. 5 SEM, EDS elemental mapping, and TEM images of control bacteria and those after CuTe treatment. (a) Control E. coli, (b) E. coli after CuTe
treatment, (c) control S. aureus, (d) S. aureus after CuTe treatment.

the overall normal structure of the control. For S. aureus, CuTe 3.5 In vivo therapeutic efficacy in intraperitoneal infection
firmly attached on bacterial surfaces in the treatment model
group (Fig. 5d) in comparison to the control (Fig. 5c), which
was confirmed by elemental mapping (EDS column in Fig. 5c Finally, the in vivo anti-bacterial effect was evaluated in an
and d). TEM images also revealed nanoparticle binding intraperitoneal infection mouse model. Decreased numbers of
on S. aureus. bacterial colonies were observed in the peritoneal fluid and

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Fig. 6 (a–e) Numbers of E. coli colonies in peritoneal fluid, major abdominal organs, and total samples in mice subjected to different treatments
(spreading was performed after dilution). (f ) Survival rates of mice in control and CuTe treated groups after intra-abdominal infection. (g) H&E stain-
ing of major organs in mice of the control group and those intraperitoneally injected with CuTe. (*P < 0.05, **P < 0.01).

major organs. Compared to the control group, CuTe therapy sig- The as-prepared CuTe mimicked grape clusters in shape and
nificantly decreased the number of E. coli colonies in the perito- acted as hunters with a high affinity for bacteria. We also preli-
neal fluid and spleen (Fig. 6a and b) and decreased the colony minarily explained the bactericidal efficacy mechanism by
numbers in the kidney and liver (Fig. 6c and d). The total revealing the activities of CuTe toward ROS generation and
amount of bacteria was also drastically reduced (Fig. 6e). More GSH depletion and demonstrating the physical association
importantly, in mice injected with a lethal E. coli dose, CuTe between this material and bacterial cells. The evidence in this
resulted in a 90% survival rate, in sharp contrast with the 10% study implies that the bacteria were killed by enhanced ROS
survival rate of the control group (Fig. 6f). Additionally, the bio- action and structural changes with cellular content leakage.
compatibility of CuTe, with respect to hemolysis and in vivo acute Given the encouraging ex vivo bacterial killing and in vivo
toxicity in major organs, was preliminary proved and demon- animal healing data, the CuTe nano-grape clusters could serve
strated in Fig. S5† and Fig. 6g, respectively. as a promising anti-bacterial agent. In this study, the CuTe
nano-grape clusters caused obvious intracellular ROS gene-
ration and leakage of nucleic acid. Such effects could possibly
4. Conclusions destroy bacterial DNA and trigger the SOS response,41 a DNA
repairing mechanism in microbes. It is worth exploring the
In conclusion, we obtained a novel anti-bacterial nano- SOS response in bacteria after incubation with CuTe nano-
material with strong intrinsic activity using a facile strategy. grape clusters. In addition, inorganic nanostructures have

This journal is © The Royal Society of Chemistry 2023 Biomater. Sci., 2023, 11, 1828–1839 | 1837
Paper Biomaterials Science

been reported to influence genes associated with inflam- 8 X. Cheng, X. Pei, W. Xie, J. Chen, Y. Li, J. Wang, H. Gao and
mation.42 The future investigation of CuTe nano-grape clusters Q. Wan, Small, 2022, 18, e2200915.
should involve evaluation of their effects on inflammation in 9 S. Mabangkhru, D. Duangthip, C. H. Chu, A. Phonghanyudh
animal models. and V. Jirarattanasopha, J. Dent., 2020, 99, 103375.
10 J. K. Schluesener and H. J. Schluesener, Arch. Toxicol.,
2013, 87, 569–576.
Author contributions 11 A. Pratsinis, P. Hervella, J. C. Leroux, S. E. Pratsinis and
G. A. Sotiriou, Small, 2013, 9, 2576–2584.
Conceptualization, Yanwen Zhou and Zhaohui Wang; formal 12 J. Liu, S. Li, Y. Fang and Z. Zhu, J. Colloid Interface Sci.,
analysis, Yanwen Zhou and Zhaohui Wang; investigation, 2019, 555, 470–479.
Yanwen Zhou and Haozhuo Lei; methodology, Yanwen Zhou, 13 G. Fang, W. Li, X. Shen, J. M. Perez-Aguilar, Y. Chong,
Haozhuo Lei, Zhaohui Wang, and Meng Wang; visualization: X. Gao, Z. Chai, C. Chen, C. Ge and R. Zhou, Nat.
Yanwen Zhou, Zhaohui Wang, and Haozhuo Lei; validation, Commun., 2018, 9, 129.
Yanwen Zhou; resources, Zhaohui Wang and Yanbin Shi; 14 J. Shan, X. Li, K. Yang, W. Xiu, Q. Wen, Y. Zhang, L. Yuwen,
supervision, Zhaohui Wang and Yanbin Shi; project adminis- L. Weng, Z. Teng and L. Wang, ACS Nano, 2019, 13, 13797–
tration, Zhaohui Wang and Yanbin Shi; funding acquisition, 13808.
Zhaohui Wang; writing–original draft, Yanwen Zhou and 15 K. Khorsandi, R. Hosseinzadeh, H. S. Esfahani, S. Keyvani-
Zhaohui Wang; writing – review and editing, Zhaohui Wang. Ghamsari and S. U. Rahman, Expert Rev. Anti-Infect. Ther.,
2021, 19, 1299–1323.
16 Y. Wang, C. Zhang, H. Zhang, L. Feng and L. Liu, Chin.
Ethical statement Chem. Lett., 2022, 33, 4605–4609.
17 F. Cao, L. Zhang, H. Wang, Y. You, Y. Wang, N. Gao, J. Ren
All animal experiments were performed in accordance with the
and X. Qu, Angew. Chem., Int. Ed., 2019, 58, 16236–16242.
requirements of Animal Management Rules of the Ministry of
18 Z. Yu, Q. Sun, W. Pan, N. Li and B. Tang, ACS Nano, 2015,
Health of People’s Republic of China and approved by Ethics
9, 11064–11074.
Committee of School of Pharmacy, Lanzhou University.
19 L. Zhang, Z. Liu, Q. Deng, Y. Sang, K. Dong, J. Ren and
X. Qu, Angew. Chem., Int. Ed., 2021, 60, 3469–3474.
20 W. Liu, H. Gu, B. Ran, W. Liu, W. Sun, D. Wang, J. Du,
Conflicts of interest J. Fan and X. Peng, Sci. China Mater., 2021, 1–10, DOI:
10.1007/s40843-021-1770-0.
There are no conflicts of interest to declare.
21 Y. Yang, X. Wu, L. Ma, C. He, S. Cao, Y. Long, J. Huang,
R. D. Rodriguez, C. Cheng, C. Zhao and L. Qiu, Adv. Mater.,
2021, 33, e2005477.
Acknowledgements 22 L. Wang, F. Gao, A. Wang, X. Chen, H. Li, X. Zhang,
We acknowledge financial support from National Natural H. Zheng, R. Ji, B. Li, X. Yu, J. Liu, Z. Gu, F. Chen and
Science Foundation of China (32000987), and Natural Science C. Chen, Adv. Mater., 2020, 32, e2005423.
Foundation of Gansu Province (20JR10RA589). 23 M. Wen, J. Ouyang, C. Wei, H. Li, W. Chen and Y. N. Liu,
Angew. Chem., Int. Ed., 2019, 58, 17425–17432.
24 G. Guo, H. Zhang, H. Shen, C. Zhu, R. He, J. Tang,
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