Dual functions of epigallocatechin gallate surface-modified Au nanorods@selenium composites for near-infrared-II light-responsive synergistic antibacterial therapy

Abstract

Diseases caused by bacterial infections pose ever-increasing threats to human health, making it important to explore alternative antibacterial strategies. Herein, epigallocatechin gallate (EGCG) surface-modified Au nanorods@selenium composites (ASE NPs) were developed for synergistic NIR-II light-responsive antibacterial therapy. In vitro antibacterial experiments demonstrated the improved antibacterial effect of ASE NPs against Staphylococcus aureus (S. aureus) compared with EGCG alone. In addition, in vivo studies demonstrated that ASE NPs cured skin wound infections and sepsis in mice caused by S. aureus. Au nanorods with excellent photothermal conversion realized synergistic photothermal therapy (PTT) in the NIR-II biowindow with an improved penetration depth at a low power density. More importantly, toxicity analysis showed that the composites had no toxic effects on major organs. Thus, the EGCG surface-modified Au nanorods@selenium composites with an NIR-II light-responsive synergistic activity hold great promise for the effective treatment of drug-resistant bacterial infections.

Keywords

Au nanorods selenium epigallocatechin gallate antibacterial photothermal effect

Introduction

Bacterial infections have become a major health problem around the world. Among them, Staphylococcus aureus (S. aureus) is a common zoonotic pathogen that can cause toxin-mediated symptoms and breast infections and is commonly responsible for mastitis.^1–4 The development of antibacterial materials has received great attention from scientists, and research on new antibacterial materials plays an important role in the development of methods to treat bacterial infections.^5,6

Au nanorods (Au NRs) have outstanding biocompatibility and unique optical properties,⁷ including horizontal and vertical surface plasmon resonance (SPR) double-spectral peaks. The vertical SPR peak position (from the visible region to the near-infrared region) depends on the aspect ratio of the rod-shaped particles, and the vertical SPR peak position can be artificially adjusted by seed-mediated method. Due to its surface SPR and other strong absorption and luminescence characteristics, under irradiation by a low-power laser, they display an excellent photothermal effect.⁸ By combining photothermal therapy (PTT) and photodynamic therapy, the optical properties of Au NRs show significant anti-tumor and antibacterial effects.^9–11 In particular, Au NRs easily react with sulfhydryl (-SH) or disulfide bonds (S–S), which facilitates their modification and provides a feasible method to functionalize materials and surfaces to improve their biological activity.^9,12,13 These studies suggest that Au NRs have promising applications for photothermal sterilization.

Selenium (Se) is an essential trace element for humans and other living beings,¹⁴ and studies have shown that in vivo, Se exists mainly in the form of various selenoproteins, which have antioxidant, anticancer, antiviral, and antifungal effects and are involved in a series of essential physiological metabolic processes in humans.^15,16 In recent years, selenium nanoparticles (Se NPs) and their complexes have been investigated and have shown tremendous potential as broad-spectrum antimicrobial agents with antibacterial activity against pathogenic bacteria, fungi, and parasites.^17–19 These nanoparticles have interesting biological activities (in vitro and in vivo), low toxicities, and excellent bioavailability.²⁰

Epigallocatechin gallate (EGCG), the major biologically active ingredient in tea polyphenols, has anti-tumor,²¹ anti-bacterial,²² anti-viral,²³ and anti-inflammatory²⁴ bioactive functions. An antimicrobial mechanism was proposed, which showed that EGCG interacts with the surface proteins of bacteria and destroys their cell membranes, thereby increasing the sensitivity of bacteria to drugs^25,26; however, monomeric EGCG has a low bioavailability because it is easily digested, metabolized, and degraded in the gastrointestinal tract. The structure of polyhydroxyl substituents also makes it difficult to transport across intestinal epithelial cells through transcellular pathways and physiological environmental factors such as pH and enzymes in the organism will also affect its stability.^27–30 To overcome this, the method of structural modification and the preparation process of the delivery body were mainly used to improve the fat solubility and stability of EGCG. EGCG was loaded onto a variety of substances, such as protein nanoparticles, polysaccharide nanoparticles, metal or non-metal nanoparticles, which prevented EGCG from isomerizing and oxidizing. This improved its sustained release and achieved its targeted delivery, which improved its biological availability, stability, and antibacterial activity.^31–34

In the absence of near-infrared light irradiation, Au NRs have no excellent antibacterial activity, it was often used to function as a drug carrier. When antibacterial drugs are modified on the surface of Au NRs, the antibacterial ability of the drugs can be enhanced, which improves both the stability of nanoparticles and their antibacterial effect.³⁵ In this paper, we wrapped Au NRs in Se NPs and then loaded EGCG onto the surface of Se NPs, which achieved synergistic antibacterial effect. The photothermal sterilization effect of Au nanorods and the antibacterial activity of Se NPs and EGCG provided a novel antimicrobial nanomaterial with improved stability. As such, these EGCG surface-modified Au nanorods@selenium nanoparticles (ASE NPs) were used to develop novel antimicrobial agents.

Materials and methods

Materials

All chemical reagents were purchased from Sigma-Aldrich (Sigma) Chemical Company. Ultrapure Luria-Bertani (LB) agar powder used to prepare the bacterial culture plates was obtained from the School of Life Sciences, Anhui Agricultural University. Escherichia coli ATCC 8739 (E. coli) and Staphylococcus aureus ATCC 6538 (S. aureus) were obtained from Anhui Agricultural University. Ultrapure water (18.25 MW) was used throughout all experiments.

Synthesis of Au NRs, Au nanorods@selenium composites, and ASE NPs

Au NRs were synthesized based on a previously reported seed-mediated method, that used a seed solution and growth solution.³⁶ The only difference between the method of this trial and the reference method was that the growth solution system was 10 times smaller (10 mL CTAB (0.1 M), 0.1 mL AgNO₃ (10 mM), 0.2 mL HAuCl₄ (25 mM), 0.04 mL HCl (1 M), with 0.07 mL of 0.0788 M ascorbic acid (VC), gently stirred), and the mixing time of the growth liquid and seed liquid was extended by 10 min. Eventually, Au NRs were collected by centrifugation (4°C, 8000 g, 10 min) and washed three times with pure water. We mixed 300 μL Na₂SeO₃ (100 mM) with 2 mL Au NRs and then added 1.2 mL of VC (100 mM) dropwise to the mixture under slow stirring for 20 min. The obtained Au nanorods@selenium composite nanoparticles (AS NPs) were washed three times with pure water.

ASE NPs were prepared as follows. Au NRs (1 mL) and 10 mg CTAB were solubilized in a water-in-ethanol solution at a constant temperature of 30°C. Pure water (5 mL) and 300 μL of 100 mM Na₂SeO₃ were added under slow stirring for 15 min. Finally, 50 mg EGCG and 10 mg VC were dissolved in the above solution until it became brown. Then, it was reacted overnight to ensure a reaction completion. During the experiment, the color gradually changed from light purple to dark brown-green.

Physical stability of ASE NPs

The colloidal stability of ASE NPs was tested by a previously reported method.³⁷ First, 0.1 mL of ASE NPs (1 mg/mL) was uniformly dispersed in 3 mL of purified water. The temperature was kept at 37°C, and the size of the ASE NPs was measured at different time intervals. The initial dimensions were compared to investigate the thermodynamic stability. The second sample was processed in the same way as the above method (37°C, standing). The transmittance of the collected sample at 1192 nm was used to indicate its kinetic stability by the same time interval and compared with the initial value. Each experiment was performed three times.

Characterization

The size and morphology of the ASE NPs were observed by transmission electron microscopy (TEM; HT7700; Hitachi, Japan; 200 kV) and scanning electron microscopy (SEM; S-4800; Hitachi, Japan). The samples were characterized by using the SEM at an accelerating potential of 15 kV. Ultraviolet–visible spectroscopy (UV-vis; Korea Scinco, Co.) and Fourier-transform infrared spectroscopy (FTIR; Nicoletteis50; Thermo Fisher Scientific USA) were used for chemical characterization. FTIR is a well-established vibrational technique used for chemical characterization of material at the molecular level with high accuracy and high spectral resolution. The sample is irradiated by infrared light of different frequencies, the molecule would absorb the radiation of specific frequencies with a ground-excited level jump occurred. The samples were blended with KBr powder and then pressed into the thin film and scanned in the range of 400–4000 cm⁻¹. Dynamic light scattering (DLS) and zeta potential measurements were used to characterize the optical properties and sizes of the ASE NPs using a Brookhaven Zeta PALS instrument. In general, zeta potentials reflect the electrostatic repulsion between dispersed particles, high value of zeta potentials represent a good dispersion of colloids particles. All measurements were conducted in triplicate at 25°C. X-ray diffraction analysis was performed using powder X-ray diffractometer (Rigaku D/max-2400, Tokyo, Japan; 45 kV; 40 mA) with Ni-filtered CuKα radiation of wavelength 1.5406 Å in the 2θ range of 10°–50° with scanning speed of 5°/min and step size 0.02°.

Bacterial culture

Escherichia coli ATCC 8739 (E. coli) and Staphylococcus aureus ATCC 6538 (S. aureus) were cultured in LB at 37°C. To obtain a logarithmic phase bacterial cell solution, the culture was inoculated in fresh medium (1:40), and the cells were incubated in a shaking incubator for 3–4 h until the solution reached an optical density of 0.5 at 600 nm.

In vitro antibacterial activity

The minimum inhibitory concentration (MIC) of different solutions was determined using a 96-well plate. The MIC values were interpreted as the lowest concentration of medicine that could inhibit bacteria. Briefly, log-phase bacteria (OD₆₀₀ = 0.5) were diluted to 1 × 10⁶ CFU/mL with fresh LB medium and then added to a 96-well plate and incubated with 0–128 μg/mL NPs solution at 37°C for 24 h. The relative viability of the bacteria was calculated by measuring the absorbance at 600 nm. The experiments were conducted in triplicate.

E. coli and S. aureus in the logarithmic growth phase were inoculated with different concentrations of EGCG, AS NPs, and ASE NPs solutions, respectively. They were inoculated in the same amount of sterile water as the negative control group, and the same concentration of ampicillin (AMP) was added as the positive control group.³⁸ The bacteria were cultured in a shaker at 37°C and 300 r/min for 12 h. After incubation, the bacterial solution was evenly spread on an LB solid medium, followed by the manual calculation of individual colonies. All experiments were carried out in triplicate.³⁹ Simultaneously, the time dependency (3–12 h) and concentration dependency (3.125–25 μg/mL) were estimated by measuring the absorbance at 600 nm.

The in vitro antibacterial activity of the synthetic material was determined by the filter paper method.⁴⁰ After the logarithmic growth phase, S. aureus and E. coli suspensions were diluted to 1.0×10⁶ CFU/mL and then evenly spread on an LB agar plate. Different concentrations of solutions (EGCG, AS NPs, and ASE NPs solutions) were equally added dropwise to dried filter paper, which was attached to the medium with sterile tweezers and pressed tightly. The diameter of the drug-sensitive tablet was 6 mm. The experiment was repeated three times, and the untreated blank medium was used as the negative control, which used the AMP as the positive control. It was incubated overnight at 37°C. The diameter of the bacteriostatic zone was measured.

Fluorescence microscopy observation (live/dead)

The log-phase bacteria (1 mL OD₆₀₀ = 0.5) were centrifuged at 3000 r/min for 5 min and washed three times with sterile water. The supernatant was discarded and re-suspended to the original volume with sterile water. Then, it was added 25.0 μg/mL of different solutions (EGCG, AS NPs, and ASE NPs solution) for 24 h. Then, a live/dead bacteria kit was used to stain the bacterial suspension (SYTO 9 and propidium iodide [PI], Life Technologies, USA). After incubating for 30 min in the dark, it was centrifuged, washed twice with sterile water, and then re-suspended. Different solutions were dropped onto tablets and observed with a fluorescence microscope. In the control group, an equal volume of sterile water was added.

Bacterial membrane integrity test

The permeability of the bacterial cell membrane was tested according to a previously reported method.⁴¹ Log-phase bacteria (OD₆₀₀ = 0.5) were inoculated with EGCG, AS NPs, or ASE NPs (25.0 μg/mL), treated at 37°C for 6 h, 12 h, 18 h, or 24 h, and then stained with PI (5 mM) for 20 min in the dark. In addition, the logarithmic phase bacteria (OD₆₀₀ = 0.5) was diluted ten-fold, 5 mM DISC3-5 was incubated in the dark for 1 h, and then 100 mM KCl was added to maintain ion balance. Then, EGCG, AS NPs, and ASE NPs (25.0 μg/mL) were added and incubated at 37°C for 6 h, 12 h, 18 h, or 24 h. Log-phase bacteria (OD₆₀₀ = 0.5) were inoculated with different concentrations of EGCG, AS NPs, or ASE NPs solutions for 12 h. The fluorescence excitation and emission wavelengths were 622 nm and 670 nm, respectively. The control group used an equal volume of water.

SEM samples were prepared using a reported method.^42,43 Three solutions (EGCG, AS NPs, and ASE NPs; 25.0 μg/mL) were, respectively, inoculated with log phase bacteria and incubated for 12 h in a shaker at 37°C and 200 r/min. Then they were centrifuged at 5000 r/min for 5 min to collect bacteria and washed three times with pure water. Subsequently, bacteria were fixed with 5% glutaraldehyde overnight and subjected to gradient alcohol dehydration. After that, the samples were dried with supercritical carbon dioxide and observed using an SEM.

Animal model

Eight-week-old male Kunming mice weighing 25 ± 1 g were used in this study.⁴⁴ Under standard environmental conditions (22 ± 3°C, 55 ± 5% humidity, and 12 h/12 h dark/light cycle), all animals were cared for in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The protocols were approved by the Committee on the Ethics of Animal Experiments at Anhui Agricultural University, Hefei, China.

Bacteremia model establishment

Briefly, eight-week-old Kunming mice (male, 25 g) were adaptively fed for 1 week and then injected with S. aureus suspension (1 × 10⁷ CFU/mL) via their tail vein. Then, the mice were given 5 mg/kg ASE NPs (once a day) via the tail vein three times, and PBS was injected into the control group. At the same time, for the in vivo photothermal treatment group, the mice were irradiated with a 1064 nm laser with a power of 1 W/cm² for 5 min. All mice were sacrificed on the ninth day, and their heart, liver, spleen, lung, and kidney were collected at the end of the experiment. Pathological analysis of major organs was also carried out by H&E staining.

Wound-healing study

Mouse models of bacterial skin infection are commonly used to assess the in vivo antimicrobial effect of drugs. Briefly, mice were intraperitoneally injected with 1% pentobarbital, and the hair on the left and right armpits was removed. After disinfection with 75% ethanol, circular skin wounds with a 2.5 cm diameter were made by incision, and the skin was removed. Then, an S. aureus suspension containing 1 × 10⁷ CFU/mL in PBS was placed in order on the left and right wound sites, respectively. ASE NPs (5 mg/kg) were injected through the tail vein every day (8 times), and PBS was injected as a control group. Using the photothermal effect of gold nanorods for local treatment, the mice were irradiated daily with a 1064 nm laser at a power of 1 W/cm² for 5 min. The images and sizes of wounds were observed and calculated. The wound tissues were collected at the end of the experiment and used for H&E staining and immunohistochemical (IHC) analysis.

In vivo toxicity test of ASE NPs

According to a method reported in the literature, we conducted an experiment to determine the toxicity of the ASE NPs in mice.⁴⁴ Kunming mice were randomly assigned to the experimental group and the control group, with three mice in each group. The experimental group was intravenously injected with a certain dose of ASE NPs solution (10 mg/kg), and the control group without medicine was set up at the same time. The body weight of the mice was recorded every day for 40 days. The mice were euthanized after the treatment, and their main organs (heart, liver, spleen, lung, and kidney) were collected. The heart, liver, spleen, lungs, and kidneys were rinsed with saline solution, fixed in 4% formaldehyde for 12 h, dehydrated, paraffin-embedded, sectioned (4 μm), and stained with hematoxylin and eosin (H&E).⁴⁵

The effects of different doses of ASE NPs on the survival of Kunming mice were also examined, and their potential toxicity was assessed. Kunming mice were injected with 5–200 mg/kg of ASE NPs solution via their tail vein over 40 days, and their survival rates were calculated. All measurements were repeated three times.

Results and discussion

Preparation and characterization of ASE NPs

Au NRs were synthesized by a seed-mediated method. Using EGCG as a surface stabilizer and VC as a reducing agent, Au was wrapped in Se spheres, and EGCG was loaded on their surface. TEM and SEM imaging showed that large amounts of uniformly dispersed Au NRs were obtained with sizes of about 50 nm (Figure 1(a) and (b)_I). Se spheres were evenly dispersed, in which the Au NRs were encapsulated (Figure 1(a) and (b)_II). As shown in Figure 1(a)_III, ASE NPs had an average diameter of 100 nm with a roughly spherical surface (Figure 1(b)_III), whose morphology and dimensions were in accordance with the TEM results. This implied that EGCG was loaded on the surface of the Se spheres. EGCG mainly acted as a surface stabilizer and reducing agent, while VC acted as a reducing agent, which caused EGCG to act as a reducing agent decreased, and the capacity of the surface stabilizer increased. The synthesis process was followed by a change in color from lavender to dark purple and finally to brownish-yellow (Figure 1(c)).

The absorbance of the synthesized NPs was using optical spectroscopy. As shown in the UV-vis spectrum (Figure 2(a)), the absorption maxima of Au NRs and Se appeared at 698 nm and 258 nm, respectively, whereas that of AS NPs appeared at 1176 nm. Additionally, the absorption maxima of EGCG appeared at 312 nm, and the maximum absorption peak of EGCG on the complex blue-shifted to 282 nm. Furthermore, after combining EGCG and Se, the maximum absorption peak of the AS NPs red-shifted from 1176 nm to 1192 nm, which indicated the inclusion of EGCG in the synthesized NPs. The chemical bonds of the ASE NPs were characterized by FT-IR. The FT-IR spectra of EGCG and ASE NPs were similar (Figure 2(b)). The EGCG spectrum contained strong and sharp peaks at 3471 cm⁻¹ (O-H stretching), indicating the presence of a large number of hydroxyl groups in EGCG. Other characteristic absorption peaks of EGCG appeared at 1697 cm⁻¹ (C = O stretching), 1614–1446 cm⁻¹ (ring stretching), 1270 cm⁻¹ (C-O stretching). The ASE NPs spectrum contained similar stretching vibration peaks. Moreover, the characteristic peaks of the functional groups in the ASE NPs spectrum displayed lower intensities than those of EGCG alone, indicating that EGCG was conjugated to the surface of ASE NPs via these functional groups.

According to Figure 2(c), the change in the transmittance of ASE NPs was less than 12% within 60 h, showing its high kinetic stability with almost no particle settling. The size of the NPs did not substantially change within 60 h because of their high thermodynamic stability. As shown in XRD spectra (Figure 2(d)), two distinct features at 2θ values of 38.17^∘ and 44.37^∘ have been observed, which can be assigned to the strongest line reflections from (111), (200) planes of the face-centered-cubic (fcc) of Au NRs (JCPDF #73-0465). In addition, the diffraction peaks at 2θ = 23.50^∘, 29.69^∘, 41.32^∘, 43.65^∘, 45.36^∘, 48.08^∘ were comparable to the (100), (101), (110), (012), (111), (200) reflections that belong to the pure hexagonal phase of selenium crystals (JCPDF #89-3697),which provided evidences that Au and Se were present. The zeta potential of the Au NPs dissolved in pure water was about 18.3 mV (Figure 2(e)), that of the AS NPs was −4.8 mV, and that of the ASE NPs was 27.6 mV. Alternating potentials demonstrated the success of each modification step. The particle size distribution of the ASE NPs was further confirmed by DLS measurements. The particles size of ASE NPs was within the range of 80–100 nm, which was in agreement with the dimensions and nanoparticle sizes estimated from TEM analysis (Figure 2(f)). The above characterizations prove that EGCG was successfully loaded on the surface of AS NPs.

In vitro antibacterial activity

The MIC value was firstly evaluated the antibacterial activity of samples, and Table 1 shows that the MIC value of ASE NPs was 82.5±0.5 μg/mL for E. coli; however, the MIC of different materials (EGCG, AS NPs, and ASE NPs) against S. aureus were significantly different, which were 55.5±0.17 μg/mL, 32.3±0.05 μg/mL, and 27.5±0.08 μg/mL for S. aureus, respectively. The antibacterial effect of the different samples toward S. aureus and E. coli was further demonstrated by measuring the average diameter of the inhibition circle. The ASE NPs showed increased anti-bacterial efficacy (growth inhibition was confirmed by the inhibitory zones) against both S. aureus compared with EGCG and AS NPs. In contrast, an antibacterial region of E. coli was not obvious. Among them, EGCG had no antibacterial activity against S. aureus and E. coli, but for ASE NPs, the sizes of the antibacterial areas were 13.2±0.15 mm and 6.5±0.02 mm for S. aureus and E. coli, respectively. Finally, compared with the standard drug AMP, ASE NPs had better antibacterial effect on S. aureus, but it had a weaker effect on E. coli, as depicted in Figure 3(a) and Table 2.

Table 1.

MIC values of S. aureus and E. coli.

Materials	S. aureus	E. coli
Materials	MIC (μg/mL)	MIC (μg/mL)
EGCG	55.5 ± 0.17	>128
AS NPs	32.3 ± 0.05	>128
ASE NPs	27.5 ± 0.08	82.5±0.5

Note: EGCG: epigallocatechin gallate; MCI: minimum inhibitory concentration; AS NPs: Au nanorods@selenium composite nanoparticles.

Table 2.

Inhibition diameter values of S. aureus and E. coli.

	S. aureus (mm)				E. coli (mm)
Concentration (μg/mL)	EGCG	AS NPs	ASE NPs	AMP	EGCG	AS NPs	ASE NPs	AMP
25	—	10.1 ± 0.05	13.2 ± 0.15	12.5 ± 0.06	—	6.1±0.03	6.5±0.02	8.3±0.04
12.5	—	8.5 ± 0.02	10.2 ± 0.04	7.1 ± 0.04	—	—	6.3±0.03	6.4±0.05
6.25	—	7.8 ± 0.05	9.5 ± 0.02	—	—	—	—	—
3.125	—	7.5 ± 0.03	8.6 ± 0.04	—	—	—	—	—

Note: EGCG: epigallocatechin gallate; MCI: minimum inhibitory concentration; AS NPs: Au nanorods@selenium composite nanoparticles; AMP: ampicillin.

The in vitro ASE NPs-induced antibacterial activity was assessed by the CFU method. As depicted in Figure 3(b), EGCG treatment did not obviously inhibit the growth of S. aureus and E. coli. In contrast, the excellent antibacterial activity was demonstrated by ASE NPs, which had a CFU value (CFU/mL) of 4.05 × 10⁷ for E. coli and 1.1 × 10⁶ for S. aureus. The survival rate of ASE NPs against S. aureus and E. coli were lower than those of the other three groups (Blank, EGCG, AS NPs). ASE NPs had better antibacterial effect on S. aureus than AMP, but its antibacterial effect on E. coli was worse than that of the standard drug AMP (Figure 3(c) and (d)). These results suggested that ASE NPs had outstanding antibacterial activities compared with raw EGCG, AS NPs, implying that the composite materials had a strong synergistic antibacterial effect. According to the above results, the antibacterial activity of ASE NPs against S. aureus was better than that against E. coli. This may be because, compared with the complex cell wall structure of E. coli, the cell wall structure of S. aureus is simple and contains a large amount of teichoic acid and is negatively charged. The positively charged ASE NPs in the solution interacted with teichoic acid and attached to the surface of bacteria, which prevented them from absorbing nutrients and excreting metabolites. This disrupted the balance of cell wall growth and the normal physiological functions of cells, thereby playing a bacteriostatic effect.⁴⁶ As such, we used S. aureus as the model organism to study the antibacterial properties of the ASE NPs. The bacterial survival rate after ASE NPs treatment was also detected by measuring the absorbance at 600 nm. According to Figure 3(e) and (f), ASE NPs treatment showed a dose- and time-dependent inhibition of S. aureus growth. The above results indicate that the ASE NPs exerted a synergistic antibacterial activity in vitro.

Fluorescence detection of the antibacterial activity

The antibacterial activity of the ASE NPs was also observed by a live/dead cell assay using SYTO@9 and PI fluorescent dye. Green/red bacterial cells were considered to be live/dead cells with intact/damaged membranes,⁴⁷ which is a direct reflection of bacterial death. Fluorescence assays were performed by 488 nm laser excitation and 530 nm emission filters for SYTO 9 (live staining). As depicted in Figure 4, most cells were alive, and the number of dead cells was minimal when treated with EGCG. After treatment with ASE NPs and AS NPs (25 μg/mL), the fluorescence intensity of SYTO@9 and PI in the cells was significantly reduced in live cells and higher in dead cells. Obviously, almost all S. aureus treated with ASE NPs (25 μg/mL) exhibited red fluorescence. There were significantly more red fluorescent bacterial cells than when treated with EGCG and AS NPs, which showed that ASE NPs had excellent bactericidal activity against S. aureus, which is consistent with the results of the previous experiment (in vitro antibacterial activity test). This may be because Gram-negative bacteria have a special outer membrane that Gram-positive bacteria lack. These outer membranes are composed of glycerophospholipids, lipopolysaccharides, and lipoproteins, which protect Gram-negative bacteria from antibacterial agents and allow them to adapt to different external environments.⁴⁸

Cell integrity studies for ASE NPs

Morphological changes of S. aureus treated with EGCG, AS NPs, and ASE NPs were observed using SEM. The SEM images (Figure 5(a)) show that untreated S. aureus maintained its membrane integrity after incubation for 12 h, and the magnified area (control) showed smooth bacterial cells with intact cell membranes. In contrast, after incubation with 25 μg/mL of materials for 12 h, both bacterial strains showed signs of wrinkling, changes in cell morphology, and damaged cell membranes. Among them, S. aureus cells treated by ASE NPs showed the greatest changes, particularly to their bacterial cell membranes. These observations confirmed that the ASE NPs interacted strongly with both Gram-positive bacterial cell membranes and caused cell death by disrupting the cell integrity.

To further explore the antibacterial mechanism, S. aureus was treated with EGCG, AS NPs, and ASE NPs for different times and concentrations, and the cells were then stained with PI and DISC3-5. PI probes can cross damaged cell membranes, bind to nucleic acids, and display bright red fluorescence. The DISC3-5 probe does not show fluorescence in intact cells, but it is released from damaged cells and displays bright green fluorescence. In short, the higher the fluorescence intensity, the more severe the damage to the bacterial cell membrane. As demonstrated in Figure 5(b) and (c), EGCG treatment did not significantly increase the red or green fluorescence, indicating that bacteria after EGCG treatment retained an intact cell membrane; however, compared with EGCG and AS NPs, ASE NPs caused greater degree, as shown by the enhanced red and green fluorescence in a time- and concentration-dependent manner, indicating that ASE NPs enhanced the destruction of bacterial cell membranes. These results also confirmed that the ASE NPs strongly bound to the cell membrane of Gram-positive bacteria and caused cell death by cell destruction.

Bacteremia model analysis

To further verify that ASE NPs exhibited a wide range of anti-infective effects, we performed rescue experiments in mice suffering from bacteremia. As demonstrated in Figure 6(a) and (b), the mice in the blank group had normal organ morphologies in all respects. In contrast, the organs of mice injected with bacteria showed signs of inflammation or lesions caused by S. aureus. Subsequently, we performed histopathological evaluation on the mouse organs. H&E staining of various organs demonstrated that S. aureus infection caused significant vacuolization, cell death of main organs, and thrombosis in the liver, accompanied by congestion. Importantly, ASE NPs treatment effectively prevented morphological changes, lesions, vacuolization, inflammation, and cell death in the major organs caused by S. aureus infection. At the same time, ASE NPs and NIR treatment further restored sites infected with S. aureus. The above results strongly indicate that ASE NPs effectively inhibited mouse bacteremia caused by S. aureus.

In vivo treatment of S. aureus-infected skin wound

Mouse models with bacterial skin infections are commonly used to evaluate the antibacterial effect of drugs in vivo. Due to the high bacteriostatic ability and no obvious toxicity of ASE NPs, an in vivo study of the treatment of skin wounds infected by S. aureus was conducted. Figure 7(a) shows macroscopic images of full-thickness wounds of mice. A near-infrared laser that matched the wavelength of the Au NRs longitudinal surface plasmon resonance (LSPR) was chosen as the light source to induce a thermal effect in Au NRs in deep subcutaneous tissues that promoted cell death.⁴⁹ The “ASE NPs + NIR” group in Figure 7(a) was used to evaluate the photothermal-based wound healing effect of Au NRs upon excitation with an 1064 nm laser. We observed that the local administration of both ASE NPs and ASE NPs with NIR inhibited wound infection and promoted healing; however, the wounds in mice in the ASE NPs + NIR group began to crust on the fifth day, and the wound area gradually decreased on the third day. After the ninth day, compared with the blank and infected groups, the treatment group displayed wounds that were nearly healed. These results illustrate that ASE NPs significantly promoted wound healing and enhanced synergistic photothermal therapy in vivo.

To explore the anti-infective effect in vivo, we assessed histopathology changes in S. aureus-infected tissue sites at the end of treatment. Then, we performed the H&E staining and assessed the expression levels of the inflammatory cytokines 1L-1β and CD116 in infected tissues by IHC. As displayed in Figure 7(b), H&E staining results show that there were many inflammatory cells in the wounds of mice infected with S. aureus. Au NRs with excellent photothermal properties realized synergistic PTT, and ASE NPs + NIR treatment in vivo reduced inflammatory cells to a greater extent. The ASE NPs-induced anti-inflammatory response was investigated by the IHC method. The increase in 1L-1β and CD116 indicated a significant induction in the inflammatory response to S. aureus infection; however, ASE NPs treatment in vivo dramatically decreased the number of inflammatory cells and inhibited the expression of 1L-1β and CD116. At the same time, the number of inflammatory cells greatly decreased, and the expression of 1L-1β and CD116 was restrained due to the photothermal effect of the Au NRs. Finally, the percentage of wound area each day was calculated by dividing the wound size of each measurement by the wound area on day 1 (Figure 7(c)). The wound area continually decreased over time. The quantitative analysis of inflammatory cells further confirmed the above conclusions (Figure 7(d)). Overall, these results demonstrate that ASE NPs realized synergistic PTT and are promising antimicrobial materials for the localized treatment of S. aureus infections.

In vivo biotoxicity studies

Drug toxicity tests are an important indicator during drug design.⁵⁰ To assess the in vivo safety of ASE NPs, we conducted physiological and histological observations of Kunming mice injected with ASE NPs. After 40 days of intravenous injection, compared with the control group, the body weight of the mice did not change significantly (Figure 8(a)). At the same time, ASE NPs at a concentration of 5 mg/kg–60 mg/kg had no toxic effect on mice (Figure 8(b)), including their vocalization, breathing, and movement. To further explore the toxicity to internal organs, the heart, liver, spleen, lungs, and kidneys were selected for histopathological assessment to determine whether S. aureus caused tissue damage, inflammation, or lesions after injection. These organs were fixed, stained, and imaged. As exhibited in Figure 8(c), histopathology by H&E staining showed that ASE NPs administration in vivo caused no significant pathological alteration to the heart, liver, spleen, lung, or kidney. In addition, no pulmonary fibrosis was identified in the lung tissue specimens. Additionally, the glomerular structure of the renal sections was normal, and no necrosis was observed in any histological specimens. The above results indicate that ASE NPs showed low toxicity and few side effects in vivo.

Conclusion

In this study, we synthesized multifunctional EGCG surface-modified Au nanorods@selenium composites to exploit the photothermal effect of Au NRs and the antibacterial activity of nano-selenium and EGCG. The characterization results showed that EGCG was loaded onto the surface of AS NPs, and ASE NPs had good dispersibility with a size range 80–100 nm. The maximum UV absorption peak of ASE NPs was red-shifted to 1192 nm. A series of experiments showed that ASE NPs had excellent thermodynamic stability and high kinetic stability. The material had an antibacterial effect on S. aureus, which was stronger than that of E. coli. The MIC values of EGCG against E. coli and S. aureus were >128 μg/mL and 55.5±0.17 μg/mL. Yet, the MIC value of ASE NPs toward E. coli was 82.5±0.5 μg/mL and 27.5±0.08 μg/mL toward S. aureus. In addition, the average diameter of the S. aureus inhibition zone of the ASE NPs was 13.2±0.15 mm. No obvious antibacterial area appeared using EGCG. The results of the CFU and inhibition zone experiments showed that the ASE NPs had a significant synergistic antibacterial activity compared with EGCG. Furthermore, a preliminary assessment of the antibacterial mechanism of the ASE NPs suggested that the ASE NPs killed the bacteria by disrupting their cell walls and membranes. In vivo experiments demonstrated that the ASE NPs significantly repaired skin wounds and cured sepsis in mice infected with S. aureus. Importantly, compared with ASE NPs, the ASE NPs irradiated with NIR successfully treated bacteremia caused by S. aureus infections in vivo. Due to their photothermal effect, ASE NPs further inhibited the inflammatory response caused by S. aureus infection and promoted wound healing, showing a synergistic anti-infection efficiency that suppressed the expression of 1L-1β and CD116. Finally, drug safety tests showed that the ASE NPs had no significant in vivo toxicity and did not cause organ damage. In short, these ASE NPs may be used as multifunctional biomedical materials with antibacterial activity and show low-power density NIR-II light-responsive synergistic activity and are the first example of next-generation antibacterial agents for industrial and clinical applications.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by The Open Fund of State Key Laboratory of Tea Plant Biology and Utilization (SKLTOF20200105), Excellent young talents support program in Colleges and Universities of Anhui Province (gxyqZD2020006), and Scientific Research Project of Anhui Education Department (KJ2019A0182).

ORCID iD

Dongdong Sun

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