Introduction

Bacterial species, as one of pathogenic agents, pose a serious threat to the human health. Over the past few decades, various antibiotics have been used in clinical practices to control bacterial infections, which has led to an intractable problem of drug resistance against pathogenic bacteria1. Hence, it is imperative to create novel antibacterial substances to address bacterial infections. In place of traditional antibiotics, photodynamic antimicrobial chemotherapy (PACT) combines light irradiation and photosensitizer agents to produce reactive oxide species (ROS) and kill pathogenic bacteria2,3,4,5,6. PACT has many advantages compared to the traditional antibiotics. It is cost-effective and exhibits high biosafety for various applications7,8,9. In PACT, various photosensitizers are used to convert incident light irradiation into oxygen radicals. As a new type of photosensitizer, Copper Cysteamine (Cu-Cy) can be activated not only by conventional UV light10, but also by X-rays11,12, microwave13 and ultrasound14 to produce singlet oxygen in deep tissue for infections. In the past, the researchers found that Cu-Cy was highly effective at killing gram-positive bacteria because the peptidoglycan layer of gram-positive bacteria was vulnerable to ROS attack. However, it showed limited antibacterial effect for gram-negative bacteria15. To enhance the antibacterial ability of Cu-Cy, we synthesized hybrids of copper cysteamine nanosheets and silver nanocluster (AgNPs) and demonstrated its superior anti-bacterial ability (Fig. 1). In this paper, the composite (Cu-Cy-PEG@AgNPs) was prepared and demonstrated excellent antibacterial activities against gram-negative and gram-positive bacteria, and in the eradication of persistent bacterial biofilms.

Fig. 1
figure 1

The synthesis of Cu-Cy-PEG@AgNPs and its anti-bacterial effect.

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Results and discussion

Synthesis and characterization of Cu-Cy-PEG@AgNPs

The synthesis of Cu-Cy-PEG@AgNPs represents an innovation based on previous reports16. Without PEG-4000, the diameter of Cu-Cy is 0.58 ± 0.01 μm. The diameter of Cu-Cy can be reduced to 180 ± 2 nm after adding PEG-4000. The added cysteamine hydrochloride serves not only for the reduction of copper chloride but also for the subsequent reduction of Ag+. The surface of Cu-Cy possesses a significant number of hydroxyl groups, which provide sites for the loading of Ag+. SEM and TEM were used to observe the morphology of this material. The Cu-Cy-PEG@AgNPs exhibited diamond-shaped nanosheets in Fig. 2a-b. It could be observed that the surface of Cu-Cy-PEG is uniformly coaded with silver nanoparticles with a particle size of about 12 nm. The EDS mapping of SEM in Fig. 2c also confirms that silver was successfully loaded on Cu-Cy-PEG nanosheets.

Fig. 2
figure 2

(a) SEM of Cu-Cy, (b) TEM and (c) EDS image of Cu-Cy-PEG@AgNPs.

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The X-ray photoelectron spectroscopy (XPS) was utilized to characterize the element change of different samples. Figure 3a shows the XPS spectra of Cu-Cy, Cu-Cy-PEG and Cu-Cy-PEG@AgNPs. The spectra confirmed the existence of Cu, C, S, O, Cl and Ag elements in the Cu-Cy-PEG@AgNPs material. As shown in Fig. 3b, the C 1s spectrum presents two binding energies of C = O (288.0 eV) and C-C (284.8 eV). Among them, C = O may be formed by the hydroxyl group under high temperature and Cu+/Ag catalysis. Figure 3c shows the N 1s profiles could be deconvoluted into a peak at 399.8 eV, which could be assigned to the NH2 bonding of cysteamine hydrochloride. For the Cu 2p spectra, as shown in Fig. 3d, six deconvoluted peaks belong to the 2p1/2 and 2p3/2 peaks for Cu+ and Cu2+, as well as the satellite peaks of copper ions. For the S 2p spectrum (Fig. 3e), two peaks were observed at binding energies of approximately 162.5 eV for Cu-S bonds and 163.7 eV for R-S-H groups respectively. As shown in Fig. 3f, the deconvoluted peak of Ag 3d5/2 was observed at 367.4 eV for the Ag 3d spectrum of pure metallic Ag. The binding energy was observed at 373.5 eV for the Ag 3d spectrum of Ag+.

Fig. 3
figure 3

(a) XPS full spectrum of Cu-Cy, Cu-Cy-PEG and Cu-Cy-PEG@AgNPs; XPS spectrum peaks of (b) C 1s, (c) N 1s, (d) Cu 2p, (e) S 2p and (f) Ag 3d of the Cu-Cy-PEG@AgNPs.

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The UV absorption spectrum of Cu-Cy series materials was observed with a peak at 365 nm, as shown in Fig. 4a. Fourier transform infrared (FTIR) spectroscopy of Cu-Cy, Cu-Cy-PEG and Cu-Cy-PEG@AgNPs is demonstrated in Fig. 4b. For Cu-Cy-PEG and Cu-Cy-PEG@AgNPs, the FTIR peaks at 3290 cm− 1 and 3282 cm− 1 could be attributed to the O-H stretching vibrations and the N-H stretching vibration of amide group, respectively. This result indicates the successful modification of PEG-4000 on Cu-Cy. The peak at 2915 cm-1 and 1604 cm-1 could be the stretching vibration peak and bending vibration peak of C-H in cysteamine hydrochloride, and the peaks at 750 ~ 1350 cm− 1 could be the C-N and C-C-N bending vibration peak of -CH2CH2NH2. These analyses confirm the successful preparation of Cu-Cy-PEG@AgNPs. Figure 4c shows the XRD spectra of Cu-Cy series materials, and the XRD pattern of Cu-Cy-PEG@AgNPs indicates higher degree of crystallinity of the sample and the oxidation states of copper, including Cu+ and Cu2+17. The XPD result of Cu-Cy-PEG@AgNPs is almost consistent with the previous literature18. Figure 4d shows the Zeta potential diagram of Cu-Cy, Cu-Cy-PEG and Cu-Cy-PEG@AgNPs. It can be seen that the potential varies from basically no charge of Cu-Cy to negative charge of modified hydroxyl group of Cu-Cy-PEG, and then to positive charge of silver nanoparticles of Cu-Cy-PEG@AgNPs, indicating the surface potential changes of materials in synthesis steps.

Fig. 4
figure 4

(a) UV-VIS spectra; (b) Fourier infrared spectra; (c) X-ray diffraction patterns of different materials; (d) Zeta potential for Cu-Cy, Cu-Cy-PEG and Cu-Cy-PEG@AgNPs; (e) Fluorescence emission spectra of SOSG after incubation with Cu-Cy Cu-Cy-PEG and Cu-Cy-PEG@AgNPs; (f) Fluorescence emission spectra of SOSG after incubation with Cu-Cy-PEG@AgNPs of different concentrations (10, 25, 50, 100 µg/mL).

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Analysis of reactive oxygen species (ROS) yield of Cu-Cy-PEG@AgNPs

When exposed to UV light, Cu-Cy materials can generate various types of ROS, including1O2 and ·OH, with 1O2 being the predominant type. The production of 1O2 in Cu-Cy, Cu-Cy-PEG, and Cu-Cy-PEG@AgNPs at equivalent concentrations was measuring fluorescence emission peaks at a wavelength of 525 nm using SOSG19. As shown in Fig. 4e, the peak of Cu-Cy-PEG@AgNPs was much stronger than the other two materials, indicating that Cu-Cy-PEG@AgNPs released more 1O2 at the same concentration. We deduce that the enhancement of the photodynamic effect of Cu-Cy-PEG@AgNPs may be caused by size reduction and loading of silver nanoparticles. Furthermore, Fig. 4f illustrates a direct correlation between fluorescence intensity and varying concentrations of Cu-Cy-PEG@AgNPs.

Antibacterial performance of Cu-Cy-PEG@AgNPs in vitro

The output of 1O2 was relevant to the antibacterial activity of Cu-Cy-PEG@AgNPs. To investigate the antibacterial properties of Cu-Cy with silver nanoparticle loading, we tested the survival ratio of the E. coli and S. aureus mixed with 25 µg/mL of Cu-Cy-PEG@AgNPs under UV irradiation. Since UV light can kill bacteria, the bacteria under UV irradiation were used as a control to evaluate the influence of UV irradiation. The bacteria viability was calculated by the ratio of the survival bacterial colonies divided by the bacteria colonies without any Cu-Cy series materials before UV irradiation. The bacterial morphology was characterized by SEM images to evaluate the anti-bacterial effect. The samples included PBS (control), PBS + UV, PBS + Cu-Cy + UV, PBS + Cu-Cy-PEG + UV and PBS + Cu-Cy-PEG@AgNPs + UV. Figure 5a-e shows the statistical viability ratio of bacteria colonies in each group and the corresponding morphology changes by SEM image. It could be noticed that the survival rate of bacteria in the “1 min UV” group approached nearly 95%, suggesting that a one-minute exposure to UV light has a marginal impact on bacterial viability. Furthermore, the SEM image depicted in Fig. 4e affirmed that this UV exposure exerted no discernible effect on the morphology of the bacteria. Supplementary Figure S1 presents the results of anti-bacterial tests conducted on Cu-Cy-PEG@AgNPs at a concentration of 32 µg/mL, with UV irradiation applied for durations of 1 min, 5 min, 10 min, 15 min, 30 min, and 60 min. The results demonstrate that just one minute of UV irradiation suffices for an effective anti-bacterial outcome.

When the bacteria were treated with Cu-Cy-PEG@AgNPs under UV exposure, E. coli and S. aureus morphological changes could be observed markedly (Fig. 5e). As shown in Fig. 4d, the red fluorescence of the dye PI was strong confirming the evident anti-bacterial effect, because PI can only enter the dead bacteria. As shown in Figure S2, Ag nanoparticles and Cu-Cy was incubated with bacteria suspension (OD600 = 0.6) under UV to compare their anti-bacterial effect, respectively, For analyzing the mechanism of anti-bacteria effect, bacteria cell walls present a negative charge and Cu-Cy-PEG@AgNPs exhibit a positive charge, so the electrostatic attraction between them is conducive for Cu-Cy-PEG@AgNPs nanomaterials to bind and interact with the bacteria cell, even to break the cell walls by generating ROS under UV irradiation. Furthermore, the antibacterial effect of the Cu-Cy-PEG@AgNPs may be enhanced by the synergistic effect of the ROS and silver nanoparticles.

Fig. 5
figure 5

Survival rates assessment of (a) E. coli and (b) S. aureus treated with different materials; (c) Flatbed assessment of E. coli and S. aureus survival; (d) Live/dead staining images of bacteria with different treatments; (e) SEM images of E. coli and S. aureus samples with different treatments.

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We also explored the influence of the concentration of Cu-Cy-PEG@AgNPs on its anti-bacteria effect. As shown in Fig. 6a-b, the survival rate of bacteria decreased with the increasing concentration of nanomaterials. When the concentration of Cu-Cy-PEG@AgNP reached 25 µg/mL, the bacteria viability of E. coli and S. aureus were zero. To verify the biosafety of the material for medical usage, mouse fibroblasts L929 shown in Fig. 6c and human breast cancer MCF-7 cell line shown in Fig. S3 were used to test the cytotoxicity of the Cu-Cy series materials. The addition of PEG and Ag nanoparticles on Cu-Cy significantly improves its biocompatibility compared to Cu-Cy materials for two cell lines. When the concentrations of Cu-Cy-PEG and Cu-Cy-PEG@AgNPs were less than 50 µg/mL, the cell viability was higher than 80%, indicating that Cu-Cy-PEG@AgNPs had a low cytotoxicity. The hemolytic test, shown in Figure S4, further confirms the improved biocompatibility of Cu-Cy-PEG@AgNPs nanomaterials. To investigate the interplay between nanomaterials and bacteria, the SEM image presented in Figure S5 revealed a tendency for the bacteria to adhere uniformly onto the planar surface of the nanomaterials. The flake-like morphology of the nanomaterials and the size of bacteria facilitated an increased contact area on the flat surface of Cu-Cy-PEG@AgNPs, thereby enhancing their mutual interaction. Under UV exposure, the nanomaterials released reactive oxygen species (ROS), which disrupted the structural integrity of microbial cell walls, cell membranes, and DNA/RNA, ultimately leading to morphological alterations in the bacteria adhered to the planar surface.

Fig. 6
figure 6

(a) The bactericidal effects of different concentrations of Cu-Cy-PEG@AgNPs on E. coli and S. aureus and (b) Bacterial viabilities; (c) Cytotoxicity of Cu-Cy, Cu-Cy-PEG and Cu-Cy-PEG@AgNPs at different concentrations (10, 25, 50, 100 µg/mL) for L929 cells.

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Antibacterial action of the Cu-Cy-PEG@AgNPs on S. Aureus biofilms

In order to assess the bacterial biofilm clearance ability of Cu-Cy series materials, different materials solutions were co-incubated with biological membranes. Syto™ 9/PI was used to stain residual live and dead bacteria on the biofilm, and the biofilm status was examined using a confocal laser scanning microscope (CLSM). As shown in Fig. 7a, it can be seen that after adding equal amounts of Cu-Cy, Cu-Cy-PEG and Cu-Cy-PEG@AgNPs, the initially dense biofilm underwent a notable reduction, transforming into a scattered and irregular appearance, accompanied by a substantial increase in the proportion of dead bacteria in the biofilm. Furthermore, we replicated the biofilm culture in 96-well plates under identical conditions. Following co-incubation with the materials, 0.1% crystal violet dye solution was introduced to quantify the number of residual bacteria. As illustrated in Fig. 7b, the dye’s color progressively faded as the residual bacterial count decreased. Figure 7c presents the corresponding statistical graph of bacterial enumeration, which aligns with previous sterilization findings. Consequently, we preliminary infer that the material’s size regulation, coupled with the incorporation of silver nanoparticles, can facilitate the penetration and removal of the biofilm’s protective layer (such as extracellular polymeric substances), thereby exposing the underlying bacteria. Once the barrier function is compromised, the weakened biofilm becomes susceptible to penetration by the released 1O2, ultimately enhancing the biofilm eradication efficiency.

Fig. 7
figure 7

(a) Confocal laser fluorescence (CLSM) images of S. aureus biofilms in different treatment groups; (b) Crystal violet staining results of S. aureus biofilm in different treatment groups and (c) proportion of residual bacteria.

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As a potent antimicrobial agent, AgNPs have found widespread application in combating bacteria, both in vitro and in vivo19. Beyond its inherent antibacterial properties, Copper cysteamine also serves as an optimal support material for the immobilization of silver nanoparticles. It provides substantial specific surface area that favors the growth and thorough dispersion of Ag nanoparticles. Consequently, the incoporation of silver nanoparticles with copper cysteamine into a nanocomposite holds considerable promise for augmenting antibacterial efficacy20,21.

Materials and methods

Materials

Coppe Chloride Dihydrate (CuCl2·2H2O, AR grade, 99.9%) was purchased from Aladdin Co., Ltd. Cysteamine hydrochloride(AR grade, 98%) were supplied by Sigm Aldrich Chemical Reagent Co., Ltd. Silver nitrate (AgNO3, AR grade, 99.8%) was purchased from Tianjin Damao Chemical Reagent Co., Ltd. Polyethylene glycol(PEG-4000, CP) and Polyvinylpyrrolidone(PVP-K30, AR grade, 99.9%) were supplied by Sinopharm Chemical Reagent Co., Ltd. Singlet oxygen fluorescence probe (SOSG) was bought from Dalian Meilun Biological Co., Ltd.

Preparation of Cu-Cy-PEG@AgNPs

In this paper, homogeneous particle size Cu-Cy-PEG@AgNPs was synthesized successfully by the in-situ reduction method22. In a 100 mL three-neck flask, 273 mg CuCl2·2H2O was added to 50 mL deionized water at 25 ℃ with a magnetic stirring at 600 rpm until dissolved. Then 32 mg PEG-4000 and 381 mg cysteamine hydrochloride was added under nitrogen protection. Then 20 mg polyvinylpyrrolidone (PVP-K30) and 2 mL AgNO3 solution (40 mM) were added and stirred for 5 min. Next, by adding sodium hydroxide solution (1 M), the pH of the solution was adjusted to weakly alkaline(pH = 7 ~ 8). After that, the solution was stirred for about 15–30 min and transferred to an oil bath at 100 °C for 20 min, and then the solution was cooled down to room temperature and centrifugated at 9000 rpm for 10 min. Finally, the solution was washed with a mixture of deionized water and ethanol (water: ethanol = 5:4) for three times, then it was put into a vacuum furnace at 40 °C overnight to obtain Cu-Cy-PEG@AgNPs.

Characterization of Cu-Cy-PEG@AgNPs

The XPS spectra were acquired using a Thermo Fisher-Nexsa instrument. The morphology of Cu-Cy-PEG@AgNPs were observed through a scanning electron microscope (FEI NovaNano 450, USA) and a transmission electron microscope (JEM-2100, Japan). Fourier transform infrared spectroscopy (FT-IR) analyses were used an equipment from Nicolet iS50. Zeta potential measurements were performed utilizing the Zetasizer Nano ZS90.

Cu-Cy-PEG@AgNPs Singlet oxygen1O2) yield analysis

Singlet oxygen fluorescence probe (SOSG) was used as a trapping agent for 1O2. The SOSG (1 × 10− 4 M) was added into the prepared Cu-Cy-PEG@AgNPs solution of different concentrations (0, 2.5, 5, 10, 25 and 50 µg/mL) with the volume ratio of 1% (v/v). After being exposed to ultraviolet light (365 nm) for 2 min, the solution was detected with a microplate reader (Synergy H4, USA).

Antibacterial property of Cu-Cy-PEG@AgNPs

The gram-negative E. coli (ACCT 25922) and gram-positive S. aureus (ATCC 25923) were obtained from Jiangsu University and selected as the representative strains to study the bacteriostatic performance of Cu-Cy-PEG@AgNPs under ultraviolet irradiation. A mixture containing 20 µL of bacterial suspension (OD600 = 0.1 or OD600 = 0.6) and 180 µL Cu-Cy-PEG@AgNPs were incubated for 5 min in a centrifuge tube with varying concentrations. Subsequently, these tubes were exposed to UV light for 1 min. Following irradiation, the bacterial suspension was diluted by a factor 104 using PBS solution. Finally, a cut bacterial suspension volume of 100 µL was spread onto LB solid medium and incubated at a temperature-controlled environment set to 37 ◦C for 24 h before counting the number of bacterial colonies formed on the medium surface. This experiment was repeated three times (Control 1: the bacterial was incubated with PBS but without UV light; Control 2: the bacterial was incubated with PBS and UV light).

Live/dead bacteria staining

Fluorescence staining technique employing propyl iodide (PI) for dead bacteria and SytoTM9 dye for live bacteria were used to assess bacterial viability under different conditions mentioned above. The collected bacterial suspensions were stained with Syto™ 9 and PI dyes for 15 min at 25 °C under dark conditions. Finally, these stained bacteria were visualized using confocal laser microscopy (Dmi8, Germany).

Cytotoxicity assay

The cytotoxicity of Cu-Cy-PEG@AgNPs towards mouse fibroblasts (L929) was assessed using the CCK-8 cell detection kit. L929 cells were inoculated into 96-well plates with a cell density of 5 × 103 per well and cultured in 1640 medium for 24 h. Subsequently, after being treated with the Cu-Cy-PEG@AgNPs series materials (0–100 µg/mL) for 24 h. Following this, each well was supplemented with 10 µL of CCK-8 solution and further incubated in cell incubator for another 2 h. Finally, the absorbance was measured at 450 nm wavelength by using an enzyme-labeled method.

Biofilm clearance test for Cu-Cy-PEG@AgNPs in vitro

The Cu-Cy series materials were dissolved in TSB medium and prepared as 100 µg/mL. 2 mL different materials solution were added into the petri dish and incubated with biofilm for 10 min. The groups which needed UV light were irradiated for 1 min. Then the materials were slowly cleaned with sterilized PBS buffer for three times. Added 1 mL of Syto™ 9/PI mixed dyes and work in the dark for 15 min, then removed the dye, using 0.85% normal saline to clear once. Finally, the biofilms were observed by laser confocal microscopy.

Conclusions

In summary, a new photodynamic antimicrobial chemotherapy photosensitizer, i.e. Cu-Cy-PEG@AgNPs, has been developed successfully. This material was demonstrated an excellent ability to release 1O2 ROS under UV and used as a photodynamic antibacterial reagent. Its antibacterial performance was evaluated against E. coli and S. aureus as model strains, and remarkable bactericidal and biofilm clearance effect were observed. Factors such as crystallite size and nanosilver loading were suggested to have a significant impact on the bactericidal performance. This work presented an essential proof for Cu-Cy-PEG@AgNPs for further photodynamic antimicrobial chemotherapy in clinical applications.