微生物学报

Samples	Cage silver(I) (1.0 mg/mL) (mL)	SCS (1.0 mg/mL) (mL)	Silver ion content (μg/mL)
Cage silver(I)	1.0	0.0	247.74
SCA1:10	1.0	10.0	20.49
SCA1:20	1.0	20.0	8.71
SCA1:30	1.0	30.0	7.84

Samples	Cage silver(I) (1.0 mg/mL) (mL)	SCS (1.0 mg/mL) (mL)	Silver ion content (μg/mL)
Cage silver(I)	1.0	0.0	247.74
SCA1:10	1.0	10.0	20.49
SCA1:20	1.0	20.0	8.71
SCA1:30	1.0	30.0	7.84

Sample	E. coli (mg/mL)
Cage silver(I)	0.031	0.12	0.015	0.031
SCS	0.620	2.50	0.620	5.00
SCA1	0.120	0.25	0.120	0.50
SCA2	0.120	0.50	0.250	1.00
SCA3	0.250	1.00	0.500	2.00

Sample	E. coli (mg/mL)
Cage silver(I)	0.031	0.12	0.015	0.031
SCS	0.620	2.50	0.620	5.00
SCA1	0.120	0.25	0.120	0.50
SCA2	0.120	0.50	0.250	1.00
SCA3	0.250	1.00	0.500	2.00

Wave length (nm)	Inhibition percentages (%)
326	SCS	8.97±2.03a	9.71±1.21a	9.98±1.12a	10.99±1.13a
SCA1	40.55±1.22c	41.90±3.12d	42.78±2.12d	43.85±3.12d
SCA2	17.81±3.34a	27.12±1.45c	28.87±2.12c	35.02±3.31c
SCA3	12.48±3.23a	13.22±1.12b	14.71±1.16b	16.26±2.12b
Cage silver(I)	37.31±1.08b	38.39±1.11d	41.36±3.14d	42.24±4.24d
517	SCS	11.54±3.26a	12.30±4.46a	13.14±2.22a	14.46±3.21a
SCA1	42.62±2.62b	43.19±3.22c	45.35±2.69d	46.47±3.26d
SCA2	17.84±4.15a	29.48±1.12b	30.32±2.23c	36.24±3.51c
SCA3	13.80±2.21a	15.11±1.21a	17.18±1.12b	20.37±1.12b
Cage silver(I)	42.34±3.92b	44.88±2.22c	52.01±3.61e	53.14±2.31e

Wave length (nm)	Inhibition percentages (%)
326	SCS	8.97±2.03a	9.71±1.21a	9.98±1.12a	10.99±1.13a
SCA1	40.55±1.22c	41.90±3.12d	42.78±2.12d	43.85±3.12d
SCA2	17.81±3.34a	27.12±1.45c	28.87±2.12c	35.02±3.31c
SCA3	12.48±3.23a	13.22±1.12b	14.71±1.16b	16.26±2.12b
Cage silver(I)	37.31±1.08b	38.39±1.11d	41.36±3.14d	42.24±4.24d
517	SCS	11.54±3.26a	12.30±4.46a	13.14±2.22a	14.46±3.21a
SCA1	42.62±2.62b	43.19±3.22c	45.35±2.69d	46.47±3.26d
SCA2	17.84±4.15a	29.48±1.12b	30.32±2.23c	36.24±3.51c
SCA3	13.80±2.21a	15.11±1.21a	17.18±1.12b	20.37±1.12b
Cage silver(I)	42.34±3.92b	44.88±2.22c	52.01±3.61e	53.14±2.31e

氮杂大环银/磺化壳聚糖络合物的抑菌性能和抗氧化活性

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周依榆 ¹^,^# , 练志峰 ¹^,^# , 吕焱 ¹ , 孙依薇 ¹ , 吴惠香 ¹ , 杨华 ¹^,² , 黄建颖 ¹^,^*

微生物学报 | 研究报告 2025,65(11): 4860-4876

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微生物学报 | 研究报告 2025, 65(11): 4860-4876

氮杂大环银/磺化壳聚糖络合物的抑菌性能和抗氧化活性

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周依榆¹^,^#, 练志峰¹^,^#, 吕焱¹, 孙依薇¹, 吴惠香¹, 杨华¹^,², 黄建颖¹^,^*

作者信息

1 浙江工商大学食品与生物工程学院，浙江杭州

2 广东海洋大学食品科学与工程学院，广东阳江

Antibacterial and antioxidant activities of Cage silver(I)/sulfonated chitosan complexes

Yiyu ZHOU¹, Zhifeng LIAN¹, Yan LÜ¹, Yiwei SUN¹, Huixiang WU¹, Hua YANG¹^,², Jianying HUANG¹^,^*

Affiliations

1 School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang, China

2 School of Food Science and Engineering, Guangdong Ocean University, Yangjiang, Guangdong, China

出版时间: 2025-11-04 doi: 10.13343/j.cnki.wsxb.20250235

文章导航

摘要

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银纳米粒子的强抗菌活性主要归因于银离子的释放，银离子通过形成Ag-S键破坏细胞膜并导致关键酶失活。目的探索银离子的固定化策略以减少其释放。方法合成了一种含氮桥头的环状配体，并将其与银离子螯合，形成氮杂大环银配合物[Cage silver(I)]，随后将其与不同比例的磺化壳聚糖(sulfonate chitosan, SCS)进行络合，得到SCS/Cage Ag(I)络合物(SCA1、SCA2、SCA3)，通过对金黄色葡萄球菌 (Staphylococcus aureus) ATCC 6538和大肠杆菌(Escherichia coli) O157:H7的最小抑菌浓度(minimum inhibitory concentration, MIC)和最小杀菌浓度(minimum bactericidal concentration, MBC)的测定、生物膜形成抑制等实验测定了它们的抗菌活性；同时通过还原能力、1,1-二苯基-2-苦肼基(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基和过氧化氢清除实验评估了它们的抗氧化性能。结果 Cage silver(I)对S. aureus ATCC 6538的MIC为0.015 mg/mL，MBC为0.031 mg/mL；对E. coli O157:H7的MIC为0.031 mg/mL，MBC为0.120 mg/mL，显示出较强的抗菌活性。通过评估DPPH自由基抑制活性、过氧化氢清除活性以及还原能力发现Cage silver(I)表现出显著的抗氧化性能，其对DPPH自由基的抑制率在326 nm和517 nm条件下分别为42.2%和53.1%。Cage silver(I)显示出较高的抗菌和抗氧化活性，其次是SCA1、SCA2、SCA3和SCS，这是由于Cage silver(I)中银离子的含量比SCA1高10倍。此外，SCA1的抗菌和抗氧化活性优于SCS，进一步表明SCS中的磺酸基团可能与银离子发生强烈配位从而产生协同效应。结论通过结合银离子和SCS的优点可以在杀菌浓度下提高药物的生物利用度，减少其在环境中的积累，并最终降低治疗成本。本研究开发的方法为增强微生物控制能力并减少环境影响提供了一种可持续的策略。

关键词

Cage / 银离子(I) / 壳聚糖 / 抗生物膜形成 / 抗氧化活性

Abstract

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The potent antibacterial activity of silver nanoparticles is primarily attributed to the release of silver ions, which disrupt cell membranes and inactivate essential enzymes through Ag-S bonding formation. Objective To explore silver ion immobilization to minimize silver release. Methods A macrocyclic cryptand with nitrogen bridgeheads was prepared and subsequently chelated with silver ions to produce Cage silver(I), which was then coordinated with different ratios of sulfonated chitosan (SCS) to form SCS/Cage Ag(I) complexes (SCA1, SCA2, and SCA3). The antioxidant activities of the complexes were assessed by reducing power and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and hydrogen peroxide scavenging assays. The antibacterial activities of the complexes were evaluated based on the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) against Staphylococcus aureus ATCC 6538 and Escherichia coli O157:H7 and the inhibition rate on biofilm formation. Results Cage silver(I) exhibited strong antibacterial activity, with the MIC of 0.015 mg/mL and MBC of 0.031 mg/mL against S. aureus ATCC 6538, and the MIC of 0.031 mg/mL and MBC of 0.120 mg/mL against E. coli O157:H7. Significant antioxidant properties of Cage silver(I) were also observed, as demonstrated by the DPPH free radical scavenging rates of 42.2% and 53.1% at 326 nm and 517 nm, respectively. Cage silver(I) exhibited the highest antibacterial and antioxidant activities, followed by SCA1, SCA2, SCA3, and SCS, because the content of silver ions in Cage silver(I) was 10-fold higher than that in SCA1. The antibacterial and antioxidant activities of SCA1 were better than those of Cage silver(I), which further indicated that the sulfonic groups of SCS may intensely coordinate with silver ions to exert synergistic effects. Conclusion Combining the merits of silver ions and SCS improves the bioavailability of the agent at microbicidal concentrations, minimizes the accumulation in the environment, and reduces treatment costs. The method developed herein offers a sustainable approach to enhance microbial control while minimizing the impact on the environment.

Key words

Cage / silver ion(I) / chitosan / antibiofilm formation / antioxidant activity

引用本文

周依榆, 练志峰, 吕焱, 孙依薇, 吴惠香, 杨华, 黄建颖. 氮杂大环银/磺化壳聚糖络合物的抑菌性能和抗氧化活性. 微生物学报, 2025 , 65 (11) : 4860 -4876 . DOI: 10.13343/j.cnki.wsxb.20250235

Yiyu ZHOU, Zhifeng LIAN, Yan LÜ, Yiwei SUN, Huixiang WU, Hua YANG, Jianying HUANG. Antibacterial and antioxidant activities of Cage silver(I)/sulfonated chitosan complexes[J]. Acta Microbiologica Sinica, 2025 , 65 (11) : 4860 -4876 . DOI: 10.13343/j.cnki.wsxb.20250235

正文

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The rapid appearance of antibiotic resistance, which creates a critical health menace worldwide, has generated renewed interest in developing products containing metals as an antibacterial agent^[1-3]. As one of the valuable and common metals, silver and its complexes have been well used as potential agents in the medical field because of their antibacterial, anti-inflammatory, and anti-neoplastic properties. Most importantly, they exhibited good antibacterial activity, including antibiotic-resistant bacteria, fungi and parasites because of their multi-site action^[1]. Metal ions are not only very rare for bacterial resistance but also possess a zombie effect^[4-5]. Furthermore, these kinds of nanomaterials encapsulated by biodegradable polymer, such as chitosan, carboxymethyl cellulose, gelatin, and polylactic acid, can offer a potential approach for long-acting sterilization and controlled delivery^[6]. Numerous functionalization of silver nanoparticles with a biocompatible biopolymer such as chitosan could improve their biological activities have been demonstrated^[1].

The metal and metal-based ions have been extensively utilized in many anti-microbial applications for decades^[7], possibly having an effect on different biochemical pathways of cells, involving in disrupting the function of normal cells, and generating irreversible damage. The high antibacterial activity of silver nanoparticles is supposed to release silver ions that destroy the cell membrane and inactive essential enzymes by Ag-S bonding^[8]. Significant bactericidal activity of silver ion assisted with ebselen against multidrug-resistant uropathogenic Escherichia coli BC1 was demonstrated by consuming total intracellular glutathione (GSH), inhibiting thioredoxin reductase activity^[9].

Subsequently, some ligands have been designed and prepared to bind with metal ions thereby regulating complex hydrophilicity or lipophilicity, stability, redox ability, as well as biological activity. Excellent antioxidant and antimicrobial activities of metals and metal-containing synthetic macrocyclic complexes allow them to have a wider range of biological applications^[10]. For example, a series of N4-acrocyclic ligands with various substituted aromatic amines have been synthesized to chelate different metal ions^[5,10]. Phenanthroline ligands complexed with transition metals [Mg(II), Cu(II), and Ag(I)] exhibited significant antibacterial activities^[11].

The medicinal inorganic chemistry of silver coordination compounds is impressively versatile, and lots of published papers highlight the novel silver complexes as promising candidates for clinical trials and subsequent therapeutic use. Nevertheless, the little toxicity of those materials is indispensable. Their large-scale application has been limited because of their residual toxicity concerns. The typically silver-coordinated small molecule compounds might remain inside the body and then react with sulfur-containing proteins which exist in the bloodstream^[4]. Therefore, it is imperative to develop an alternative approach for immobilizing silver ions into biomacromolecules to enhance antibacterial activity and mitigate cytotoxic effects. As a kind of anion biopolymer, sulfonate chitosan (SCS) with good water solubility, biocompatibility, biodegradability, antibacterial activity, and coordination potential makes its potential application in the biomedical fields^[12]. Enhanced antibacterial properties, stability and low cytotoxicity were found after metal ions coordination with chitosan and its derivatives^[5,13], such as AgBrNPs@copolymer-decorated CS^[14], AgNPs@CS/SCS)^[15], and cryptate copper(II)/SCS^[5]. In this context, a new class of low-cost, easily synthesizable N4-acrocyclic ligand was prepared. After coordination with silver(I) followed by complexing with different ratios of SCS, their antioxidant and antimicrobial activities were investigated. The novelty of Cage silver(I)/SCS complexes developed herein may be fundamental in the selection of new metallo-drugs with very little or no toxicity to humans.

1 Materials and Methods

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1.1 Materials and microorganisms

Chitosan (CS) with a deacetylation degree of 91% and a centipoise viscosity of 60 mPa·s (1.0%, 20 ℃) was afforded by Qingdao Honghai Biotechnology Company (Shandong, China). LIVE/DEAD^® BacLight™ Bacterial Viability Kit for microscopy was bought from ThermoFisher Scientific (Shanghai, China). Other chemical reagents and solvents were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China) and were used directly without further purification.

1.2 Microorganism cultivation

The tested bacteria strains Staphylococcus aureus ATCC 6538 and Escherichia coli O157:H7 were bought from the China General Microbiological Culture (CGMCC, Beijing, China). Strainswere respectively grown in Luria-Bertani (LB, Land Bridge, China) for E. coli and Trypticase-Soy-Broth (TSB, Land Bridge, China) supplemented with 1.0% glucose (Macklin, China) for S. aureus. All samples were incubated at 37 ℃ in a shaking incubator for 24 h, and the bacterial concentration was determined with a microplate reader (Tecan, Switzerland) at optical density (OD) of 600 nm (OD₆₀₀) of 1.0 corresponded to a concentration of 10⁹ colony-forming units per-milliliter (CFU/mL)^[16].

1.3 Apparatus and characterization

The ultraviolet-visible (UV-vis) spectra were measured with a spectrophotometer (Shimadzu, Japan) at room temperature (RT). Fourier transform infrared spectra (FT-IR) were recorded using a spectrometer (ThermoFisher Scientific, USA). High resolution electrospray ionization mass spectroscopy (HRMS) was determined on a TRAP instrument (A&B, USA). ¹H NMR spectra were recorded on a 500 MHz Bruker Avance NMR spectrometer (Bruker, Switzerland) at 25 °C. Silver ion concentrations were measured with a 2% nitric acid digestion method using an inductively coupled plasma mass spectrometer (ICP-MS, Agilent).

1.3.1 Synthesis of SCS^[17] and Cage silver(I)

CS (1.0 g) was added to 80.0 mL of 2.0% aqueous acetic acid solution with stirring until completely dissolved. After 2.0 g of 1,3-propane sultone addition, the resulting mixture was further stirred for 6 h at 60 ℃ under a nitrogen atmosphere. After that, the solution was poured into cold acetone with vigorous stirring to precipitate white product. The white solid was washed with methylene chloride to remove unreacted 1,3-propane sultone and was vacuum-dried for 10 h.

The dinuclear silver complex was prepared according to the previous literature^[18], which was illustrated in Figure 1. 0.48 g of benzene-1,3-dicarboxaldehyde (3.60 mmol) was dissolved in 90.0 mL of acetonitrile (MeCN) solution. Then 0.37 g of tris(2-aminoethyl)amine (2.50 mmol) in 30.0 mL of MeCN was added dropwise with gently stirring for 10 h at RT. The resulting imine 1 was obtained (0.53 g, yield 75.5%) after filtration. Next, sodium borohydride (0.27 g, 7.00 mmol) was mixed with imines (0.18 g, 0.03 mmol), which were dissolved in 10.0 mL of methanol, and the mixture was stirred and refluxed overnight. The reaction completion was monitored by monitoring with thin-layer chromatography (TLC). After removal of the solvent, the residue was suspended in 12.0 mL of 2.00 mol/L ammonium chloride solution, then extracted with dichloromethane (12.0 mL) three times, and subsequently dried with Na₂SO₄. The resulting organic phases were evaporated under vacuum to afford Cage 2 (0.12 g, yield 69.2%).

Cage 2 (227.5 mg, 0.38 mmol) was dissolved in 40.0 mL of degassed MeCN/MeOH (4:1) followed by adding 132.5 mg of AgNO₃ (0.78 mmol). The mixture was stirred at RT for 10 h. After the removal of the solvent under reduced pressure, the residue was re-dissolved with 5.0 mL of MeCN. The resulting Cage silver(I) was precipitated by Et₂O addition (78.2 mg, yield 25.3%).

1.3.2 Preparation of SCS/Cage silver(I) (SCA) complex

Cage silver(I) was dissolved in DMSO at 1.0% (W/V) concentration in the final solution, while SCS was dissolved in Millipore water (Merck Millipore, Germany) with stirring at RT till complete dissolution. Then they were mixed with different weight ratios to afford SCS/Cage silver(I) 10:1 (SCA1), SCS/Cage silver(I) 20:1 (SCA2), and SCS/Cage silver(I) 30:1 (SCA3).

1.4 Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) determination

MIC was determined by using the microtiter broth dilution method^[12]. LB and TSB supplemented with 1.0% glucose were used as the negative controls, while strains with corresponding LB and TSB were served as the positive controls. An initial concentration of 4.0 mg/mL of SCA1, SCA2, and SCA3 was serially diluted to 0.015 625, 0.031 25, 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0, and 4.0 mg/mL, and SCS was from 0.039 062 5 to 10.0 mg/mL, while the concentrations of Cage Ag ranged from 0.007 812 5 to 0.5 mg/mL. 100 μL of each solution was added to 96-well plates, followed by the addition of 100 μL of bacterial suspension (10⁶ CFU/mL). All inoculated microplates were incubated at 37 ℃ for 24 h, followed by the absorbance values read at OD₆₀₀ with a microplate reader (Tecan, Switzerland). The MIC was determined as the lowest concentration that showed complete inhibition of visible growth.

Tryptic-Soy-Agar (TSA, Land Bridge, China) plates were applied for MBC value determination according to the number of colony-forming units^[12]. All samples were performed in triplicate.

1.5 Biofilm formation inhibition

1.5.1 Adhesion assay

The effects of all samples on the biofilm formation were determined according to the previous literature^[12]. Briefly, the activated E. coli and S. aureus suspensions (10⁸ CFU/mL) were separately pre-mixed with 10.0 mL of the test compound solutions to achieve final compound concentrations ranging from 1MIC concentration to 1/4MIC concentration. The sterilized 1 cm²×1 cm² cover glass was pre-placed in well plates, then an aliquot of 2.0 mL of the cell/compound mixtures was added for replicate testing. After 48 h of biofilm formation in the 24-well plates, the cover glass was gently removed from the plates and subsequently rinsed with sterile phosphate buffer saline (PBS, 0.01 mol/L, pH 7.4) to remove non-adherent cells. Then, the rinsed cover glass was placed in a centrifuge tube containing 2.0 mL normal saline, and ultrasound was performed for 5 min at 150 W power. Followed by shaking with a vortex oscillator for 1 min, 100.0 μL of the oscillating bacterial solution was added to TSA plates for general colony count.

1.5.2 Confocal laser scanning microscopy (CLSM)^[5]

The E. coli and S. aureus cells treated with or without different samples were grown in a confocal FluroDish petri dish with TSB (1.0% glucose, 3.0 mL), and then 1.0% suspension (10⁷ CFU/mL) of stains was separately added to the final concentrations of the samples were 1MIC. Sterilized deionized water without samples was used as a control. After 48 hours incubation at 37 ℃, the planktonic bacteria on the surface of the plate was gently rinsed with sterile PBS (pH 7.4). Then, all bacterial cells were stained with SYTO9 and propidium iodide (PI), the viability of corresponding cells was assessed by using a LIVE/DEAD^® Bacterial Staining Kit (ThermoFisher Scientific), and the distribution of live and dead bacteria in biofilms were observed by a CLSM (Zeiss, Germany).

1.6 DPPH radical scavenging activity

A stable free radical DPPH was used to evaluate the scavenging effect of all resulting complexes according to the previous reports^[19-20]. 1.0 mL of each different concentrations sample solution was separately mixed with 9.0 mL of ethanol solution of DPPH (200.0 μmol/L) to afford a solution of DPPH with a final concentration of 100.0 μmol/L. The mixture was reacted in a water bath at 25 °C for 30 min, then the absorbance of the resulting reaction was read from 200 nm to 800 nm by a spectrophotometer (Shimadzu, Japan). The DPPH radical scavenging activity was expressed as a percentage of inhibition activity according to Equation (1).

I n h i b i t i o n = (A 0 - A X) ∕ A 0 × 100 %

(1)

where A₀ is the absorbance of the control and A_X is the absorbance of different concentrations samples. All samples were measured in triplicate.

1.7 Reducing power assay^[19]

1.0 mL of each different concentrations sample solutions ranging from 0 mg/mL to 4.0 mg/mL were respectively mixed with 0.2 mL of fresh PBS (0.2 mol/L, pH 6.6) and 1.0 mL of 1.0% potassium ferricyanide. The mixtures were stirred at 50 ℃ for 20 min, respectively. After 1.0 mL of 10% trichloroacetic acid addition, the resulting reaction mixtures were centrifuged at 6 000 r/min for 10 min (Cence). 2.0 mL of each supernatant was collected and added with 0.35 mL of 3.0% ferric chloride solution and 1.0 mL of distilled water, followed by the absorbance that read at 700 nm. Reducing power can be evaluated by the increased absorbance of the reaction mixture. All samples were assayed in triplicate.

1.8 Scavenging of hydrogen peroxide^[21]

The sample solution (4.0 mg/mL) was mixed with 2.4 mL of 0.1 mol/L PBS (pH 7.4), followed by adding 0.6 mL of 43.0 mmol/L solution of hydrogen peroxide. The absorbance was measured at 230 nm from 0 min to 40 min and at every 5 min interval until 240 min. While a separate blank without hydrogen peroxide solution was served as background subtraction. The radical scavenging ability was expressed as a percentage of scavenging activity against hydrogen peroxide according to Equation (2).

I n h i b i t i o n = 1 - (A 0 - A X) / (A 0' - A X') × 100 %

(2)

where A₀ and A₀′ are the absorbance read at the initial incubation time for the sample and control, respectively, and A_Xand A_X′ are the absorbance of sample and control read at successive time, respectively. All samples were assayed in triplicate.

1.9 Statistical analysis

Analysis of variance (ANOVA) (P<0.05) was performed using SPSS 27 software. The display data was expressed as the means±SD. All experiments were performed at least three times. Differences at P<0.05 were considered to be statistically significant.

2 Results

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2.1 Characterization

As illustrated in Figure 1, Cage silver(I) was prepared using benzene-1,3-dicarboxaldehyde and tris(2-aminoethyl)amine as starting materials, followed by a reduction reaction using sodium borohydride as a reducing agent, and subsequently coordinated with silver ions. Its structure was characterized by ¹H NMR and HRMS (high-resolution mass spectroscopy) spectra. The ¹H NMR spectra of Cage and Cage silver(I) were presented in Figure 2, which confirmed their formation.

The structure of the Cage silver(I) complex was further supported by HRMS. The ESI-MS of Cage silver(I) (Figure 3) showed related peaks at m/z=407.130 4 and m/z=705.354 1, corresponding to [M_cage+2Ag]²⁺ and [M_cage+Ag]⁺, respectively. These two peaks were isotopically resolved and agreed very well with their calculated isotopic distributions.

The silver ion concentrations of each sample analyzed by ICP-MS were 247.74, 20.49, 8.71, and 7.84 μg/mL corresponding to Cage silver(I), SCA1, SCA2, and SCA3 (Table 1).

To further illustrate the structure and interaction between functional groups of Cage silver(I), SCS, and formation of SCA, all ground and dried samples were examined in the infrared range (4 000 cm^-1 to 400 cm^-1) by a FT-IR spectrophotometer, and the results were shown in Figure 4. FT-IR spectrum of SCS showed the typical stretching vibration peaks of secondary N-H and O-H of SCS appeared around 3 440 cm^-1, N-H stretching vibration at 1 646 cm^-1 was attributed to the acetylated units of chitosan, S=O stretching vibration and O=S=O symmetric vibration of sulfonic acid group were at 1 180 cm^-1 and 1 030 cm^-1(Figure 4A) which was following the previous report^[11].

As to Cage (Figure 4C), the bands appeared at 2 945 cm^-1 and 2 821 cm^-1 accounted for C-H stretching vibration of methylene, and at 1 451 cm^-1 was C-H bending vibration band of methylene. The present of the band at 3 030 cm^-1 was supposed to the C-H stretching vibration of the benzene ring. While the bands at 803 cm^-1 and 711 cm^-1 are attributed to the bending vibration of C-H in the benzene ring. The band at 1 615 cm^-1 corresponded to the C=C stretching vibration of the benzene ring. After chelator with silver ions, the N-H vibrational bands of Cage at 3 431 cm^-1 shifted to 3 449 cm^-1, The sharp peak appeared at 3 266 cm^-1 possibly ascribed to the stretching vibration of C=C-H of benzene, while sharp peak around 2 839 cm^-1 was C-H vibration of Cage silver(I). When Cage silver(I) complexed with SCS, the typical secondary N-H stretching vibration peak at 3 426 cm^-1, C=C-H of benzene at 3 226 cm^-1, and N-H bend near 1 647 cm^-1 ascribed to amine groups and acetylated units of chitosan could be seen from the FT-IR spectrum of SCA1 (Figure 4B). The decrease in peak intensity of Cage silver(I) at 1 358 cm^-1 was evidence of the Cage silver(I) complexed with SCS.

2.2 Antibacterial activity

The antibacterial activities of all samples against S. aureus, and E. coli were investigated by MIC and MBC determination, while methicillin against S. aureus and polymyxin B against E. coli were used as controls. As presented in Table 2 and Figure 5, the growth of S. aureus and E. coli was inhibited by Cage silver(I) at 0.015 mg/mL and 0.031 mg/mL, with the corresponding MBC values 0.031 mg/mL and 0.120 mg/mL, respectively. Apparently, the antibacterial activities of all tested samples gradually reduced when the content of Cage silver(I) decreased in SCA complexes, indicating it played an important role in the bacteriostatic capability of SCA. The MIC and MBC of SCA1 were 0.12 mg/mL and 0.50 mg/mL against S. aureus,0.12 mg/mL and 0.25 mg/mLagainst E. coli, exhibiting stronger antibacterial activities than those of SCS with 0.62 mg/mL and 5.00 mg/mL against S. aureus,0.62 mg/mL and 2.50 mg/mLagainst E. coli, which was consistent with a previous study^[22].

2.3 Antiadhesion and antibiofilm formation

All samples exhibited adhesion inhibitory properties against E. coli and S. aureus with Cage silver(I) content-dependent mode (Figure 6). As expected, Cage silver(I) showed the best anti-adhesion among all tested samples. The colony forming units of E. coli and S. aureus were 3.90 lg CFU/cm² and 4.16 lg CFU/cm² when 1MIC Cage silver(I) treated, followed by SCA1 (4.60 lg CFU/cm² and 4.45 lg CFU/cm²), SCA2 (4.98 lg CFU/cm² and 5.14 lg CFU/cm²), SCA3 (4.89 lg CFU/cm² and 5.75 lg CFU/cm²), SCS (5.34 lg CFU/cm² and 5.70 lg CFU/cm²), and the control (8.44 lg CFU/cm² and 8.69 lg CFU/cm²). It is found that S. aureus was more susceptible to Cage silver(I) than E. coli,whileSCA1, SCA2, SCA3, and SCS exhibited better anti-adhesion activities against E. coli than against S. aureus.

2.4 CLSM

As shown in Figure 7A, the distribution of S. aureus biofilm in the control group was dense and uniform with good cell activity. Less green fluorescence intensity and more reddish fluorescent cells (Figure 7B) were found compared with the control in the confocal microscopy imaging of S. aureus treated with SCS. Importantly, rare fluorescence intensity was observed in Cage silver(I) treated S. aureus (Figure 7D),followed by SCA1 treatment(Figure 7C). Similar effects of the tested samples against E. coli biofilm can be seen from Figure 8A-8D, indicating that Cage silver(I) exhibited the strongest anti-biofilm formation activity against E. coli and S. aureus among SCA1 and SCS at MIC concentration.

Dramatic reduction of the cell viability and density illustrated that Cage silver(I) could inhibit the adhesion of S. aureus and E. coli to the coverslip surfaces, which was followed by SCA1 and SCS. These results were consistent with the results of the bacterial biofilm adhesion (Figure 6).

2.5 DPPH radical scavenging determination

DPPH, a semi-stable free radical, is a radical scavenger because it is capable of accepting electrons from reactive radicals. The mechanism of DPPH free radical scavenging may be very complex because the DPPH molecules cannot only react with electrophile reagents but hydroxyl radicals, indicating that the absorbance of DPPH absorption bands at 517 nm and 326 nm, possibly change simultaneously (Figure 9A). Therefore, the determination of radical scavenging ability of compounds was recorded based on photobleaching of DPPH at 517 nm and 326 nm is reasonable^[19].

Figure 9 showed a set of UV-vis spectra measured upon DPPH solution reacting with various samples. It can be clearly seen that different absorbance decreases of DPPH absorption bands at 517 nm and 326 nm occurred with a concentration-dependent mode in the DPPH radical scavenging activity assessment of all tested samples (Figure 9 and Table 3).

A substantial and statistically significant decrease in the DPPH levels of all samples can be seen from Table 3. Cage silver(I) exhibited the strongest antioxidant activity among all tested samples, and its inhibition percentages of DPPH radicals reached 42.2% and 53.1%, respectively, based on the photobleaching of DPPH at 326 nm and 517 nm. The inhibitory activities of SCA1, SCA2, and SCA3 were 43.9%, 35.0%, 16.3% at 326 nm, and 46.4%, 36.2%, 20.3% at 517 nm at the concentration of 0.4 mg/mL. While the inhibitory activity of SCS against DPPH was 11.0% and 14.5% based on photobleaching of DPPH at 326 nm and at 517 nm when the concentration of SCS was 0.4 mg/mL.

2.6 Reducing power

As shown in Figure 9, the reducing power of all samples was in concentration-dependent manner. Significant reducing power of Cage silver(I) could be observed from Figure 9. When the concentration was 0.4 mg/mL, the reducing power of Cage silver(I) was up to 1.85. As expected, the reducing power was reduced in order of SCA1, SCA2, and SCA3, which were 0.58, 0.29 and 0.27 when the concentration of samples was 0.4 mg/mL, while the reducing power of SCS was 0.09 at 0.4 mg/mL. The antioxidant capacity of SCS was due to their hydrogen-donating ability^[23]. However, the maximum absorbance was observed for Cage silver(I) which was supposed to the presence of nitrogen and silver atoms in Cage silver(I).

2.7 Scavenging activity of hydrogen peroxide

As shown in Figure 10, the scavenging activity of samples was directly correlated with the reaction time, which reached 57.3%, 49.8%, 38.9%, and 37.4% of SCA1, SCA2, SCA3, and SCS, respectively, in 40 min reaction. The scavenging ability of Cage silver(I) against hydrogen peroxide was not shown herein because high concentrations of silver ions in Cage silver(I) reacted with hydrogen peroxide to produce silver oxide^[19].

3 Discussion

收起

Silver is less toxic to human cells, however, a growing environmental concern in the increasing silver level accumulation after long intensity of exposure will produce residual toxicity and ultimately be harmful to public health. Herein, N4-acrocyclic ligand (Cage2) was successfully synthesized, and was confirmed by ¹H NMR spectra which were following the literature^[18]. Then the silver ions were tightly chelated in the nitrogen macrocyclic and further mixed with the SCS, addressing new materials which can alleviate silver release.

After structure characterization by ¹H NMR, HRMS, and FT-IR, the antibacterial and antioxidant activities of prepared Cage silver(I), and SCA were studied. As expected, Cage silver(I) exhibited excellent antibacterial activities against S. aureus and E. coli with the lowest MIC and MBC values (0.015 mg/mL and 0.031 mg/mL), followed by SCA1, SCA2, SCA3, and SCS, indicating that the content of Cage silver(I) played an important role in bacteriostatic capability of SCA. Similar trend of antibiofilm formation ability present in CLSM further demonstrated the significantly important Cage silver(I) content in SCA. These results are in accordance with the previous report^[5]. While the MIC values of methicillin against S. aureus and polymyxin B against E. coli with 0.76 mg/L and 2.60 mg/L^[16], respectively, show slightly better than Cage silver(I), however, both are easy to develop resistance, and the resistance development of SCA is worthy to be assessed.

Additionally, commercially available silver-based disinfectants such as silver nitrate^[24] and nano-silver particles^[25] exhibited antibacterial activities with the MIC values ranging from 0.01 mg/mL to 0.04 mg/mL for silver nitrate against S. aureus and E. coli, and 12.5 mg/mL to 25.0 mg/mL for nano-silver particles against S. aureus and E. coli, further demonstrated that silver ions played a key bactericidal role, which was following the previous study^[26]. Herein, the MICs of Cage silver(I) and SCA1 against S. aureus and E. coli were 0.015 mg/mL and 0.12 mg/mL, indicative of their significant inhibitory activities. The mode of antimicrobial action of silver ions is supposed to bind to the surface of the bacterial cell membrane and subsequently enter into cell to interfere with the activity of essential intracellular enzymes and DNA, causing enzyme degradation, cell protein inactivation, and DNA breakage, eventually resulted in the bacteria’s death^[2,27]. Evidence also suggests that the antimicrobial activity of silver ions may be related to the redox reaction which impairs the cell cytoplasm, leading to inhibition of cell replication and cell death^[7,27]. Therefore, the efficiency of the antibacterial activity of silver complex depends upon the content of silver ions, which were exposed to the bacteria^[7]. Both chemisorbed Ag⁺ and AgNP aggregation would influence their antibacterial efficacy^[28], however, AgNP and silver ions can easily migrate to the environment, ultimately threatening human health.

In our study, the silver ions bound to the four nitrogen atoms at the trend end of the macro-bicyclic cryptand, making the coordination geometry very distorted, in which all four nitrogen atoms were on one side of the metal ion which has been reported by Ray and Bharadwaj^[29]. Importantly, the spherically symmetric configuration of the silver(I) ion allows the coordination number to the Cage is 2 which was proved by ESI-MS. All these factors influenced both the electronic and steric properties of Cage silver(I), which in turn modulated the rate of silver release, also maintaining their bioavailability over an extended period and preventing reinfection or resistance. The proposed antibacterial mechanism of SCA is that silver and SCS can bind bacterial cell membrane, destroy the cell wall, and induce oxidative damage of lipid and protein present in cells.

Additionally, overproduction of activated oxygen species is one of the major contributors to oxidative damage of DNA, lipids and proteins, which increases the chance of cancer, aging, inflammation, as well as cardiovascular diseases. Many transition metal complexes also showed significant antioxidant activities^[30]. The N4-acrocyclic ligand synthesized in our study had the ability to chelate with silver ions, subsequently complex with SCS. Herein, significant antioxidant activities of Cage silver(I) and SCA were demonstrated by the reducing power, DPPH radical, and hydrogen peroxide scavenging assays (Table 3, Figure 9, and Figure 10).

Based on the antibacterial and antioxidant activities assessment, Cage silver(I) exhibited the highest antibacterial and antioxidant activity followed by SCA1, SCA2, SCA3, and SCS, which was because the content of silver ions in Cage silver(I) was 10-fold higher than that of SCA1 (Table 1). While the antibacterial and antioxidant activity of SCA1 was less than 10-fold those of Cage silver(I), further indicating that the amino sulfonic groups of SCS may be intensely coordinated with silver ions, which would be helpful for synergistic effects. The potent antibacterial and antioxidant activities of SCA were in agreement with the results of polysaccharide-based silver nanoparticles and plant extract-based silver nanoparticles^[31-33].

4 Conclusion

收起

In this study, Cage silver(I) was prepared and complexed with SCS. After characterization by NMR, FT-IR, HRMS, and ICP-MS analysis, the antibacterial activity and antioxidant capacity assay of all samples were investigated. The mass ratio of 10:1 between SCS and Cage silver(I) was considered as the optimal ratio by determining the antibacterial activities against S. aureus and E. coli. Also, the study discovered that the complex of SCS and Cage silver(I) exhibited a remarkable ability to counteract free radicals as an antioxidant, according to the scavenging capacities against DPPH, reducing power and scavenging of hydrogen peroxide. The chitosan matrix not only exerted synergistic effects but also mitigated the toxicity produced by silver ion release and ultimately reduced environmental accumulation. Notably, chitosan is inexpensive, while the minimal content of silver ions in SCA1 (20.49 μg/mL) lowers the metal silver cost, making it suitable for scale-up production and application. Additionally, the Cage synthesis involves Schiff base reaction and reduction, which are easy to handle, and the starting materials are cheap. In the future, the detailed mechanisms of action, long-term safety, stability, and efficacy in vivo and in vitro will be studied to develop a novel antimicrobial agent using this immobilization strategy.

Credit authorship contribution statement

收起

ZHOU Yiyu: Formal analysis, investigation, writing-original draft preparation; LIAN Zhifeng: Formal analysis, investigation, writing-original draft preparation; LÜ Yan, SUN Yiwei, and WU Huixiang: Software, validation; YANG Hua and HUANG Jianying: Conceptualization, writing-reviewing and editing.

基金

收起

浙江省自然科学基金(LY20B040001)

参考文献

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doi: 10.13343/j.cnki.wsxb.20250235

接收时间：2025-02-20
首发时间：2025-11-10
出版时间：2025-11-04

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收稿日期：2025-02-20
录用日期：2025-04-10

基金

Zhejiang Provincial Natural Science Foundation(LY20B040001)

浙江省自然科学基金(LY20B040001)

作者信息

1 浙江工商大学食品与生物工程学院，浙江杭州

2 广东海洋大学食品科学与工程学院，广东阳江

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Samples	Cage silver(I) (1.0 mg/mL) (mL)	SCS (1.0 mg/mL) (mL)	Silver ion content (μg/mL)
Cage silver(I)	1.0	0.0	247.74
SCA1:10	1.0	10.0	20.49
SCA1:20	1.0	20.0	8.71
SCA1:30	1.0	30.0	7.84

Samples

Cage silver(I) (1.0 mg/mL) (mL)

SCS (1.0 mg/mL) (mL)

Silver ion content (μg/mL)

Cage silver(I)

1.0

0.0

247.74

SCA1:10

1.0

10.0

20.49

SCA1:20

1.0

20.0

8.71

SCA1:30

1.0

30.0

7.84

Sample	E. coli (mg/mL)
Cage silver(I)	0.031	0.12	0.015	0.031
SCS	0.620	2.50	0.620	5.00
SCA1	0.120	0.25	0.120	0.50
SCA2	0.120	0.50	0.250	1.00
SCA3	0.250	1.00	0.500	2.00

Sample

E. coli (mg/mL)

S. aureus (mg/mL)

MIC

MBC

MIC

MBC

Cage silver(I)

0.031

0.12

0.015

0.031

SCS

0.620

2.50

0.620

5.00

SCA1

0.120

0.25

0.120

0.50

SCA2

0.120

0.50

0.250

1.00

SCA3

0.250

1.00

0.500

2.00

Wave length (nm)	Inhibition percentages (%)
326	SCS	8.97±2.03a	9.71±1.21a	9.98±1.12a	10.99±1.13a
SCA1	40.55±1.22c	41.90±3.12d	42.78±2.12d	43.85±3.12d
SCA2	17.81±3.34a	27.12±1.45c	28.87±2.12c	35.02±3.31c
SCA3	12.48±3.23a	13.22±1.12b	14.71±1.16b	16.26±2.12b
Cage silver(I)	37.31±1.08b	38.39±1.11d	41.36±3.14d	42.24±4.24d
517	SCS	11.54±3.26a	12.30±4.46a	13.14±2.22a	14.46±3.21a
SCA1	42.62±2.62b	43.19±3.22c	45.35±2.69d	46.47±3.26d
SCA2	17.84±4.15a	29.48±1.12b	30.32±2.23c	36.24±3.51c
SCA3	13.80±2.21a	15.11±1.21a	17.18±1.12b	20.37±1.12b
Cage silver(I)	42.34±3.92b	44.88±2.22c	52.01±3.61e	53.14±2.31e

Wave

length (nm)

Inhibition percentages (%)

Sample

0.1 mg/mL

0.2 mg/mL

0.3 mg/mL

0.4 mg/mL

326

SCS

8.97±2.03a

9.71±1.21a

9.98±1.12a

10.99±1.13a

SCA1

40.55±1.22c

41.90±3.12d

42.78±2.12d

43.85±3.12d

SCA2

17.81±3.34a

27.12±1.45c

28.87±2.12c

35.02±3.31c

SCA3

12.48±3.23a

13.22±1.12b

14.71±1.16b

16.26±2.12b

Cage silver(I)

37.31±1.08b

38.39±1.11d

41.36±3.14d

42.24±4.24d

517

SCS

11.54±3.26a

12.30±4.46a

13.14±2.22a

14.46±3.21a

SCA1

42.62±2.62b

43.19±3.22c

45.35±2.69d

46.47±3.26d

SCA2

17.84±4.15a

29.48±1.12b

30.32±2.23c

36.24±3.51c

SCA3

13.80±2.21a

15.11±1.21a

17.18±1.12b

20.37±1.12b

Cage silver(I)

42.34±3.92b

44.88±2.22c

52.01±3.61e

53.14±2.31e