Facile synthesis of silver nanoparticles deposited on a calcium silicate hydrate composite as an efficient bactericidal agent

Abstract

Silver nanoparticles (Ag NPs) have been widely used for the disinfection and prevention of pathogenic bacteria. However, the aggregation and instability of Ag NPs significantly limit their bactericidal activity. As an alternative method, stabilization of Ag NPs on an inorganic biomaterial is a promising strategy. In this study, an inorganic material with good biocompatibility and bioactivity, calcium silicate hydrate (CSH), was employed as the host substance. A facile and efficient method was proposed to produce Ag NPs deposited on a CSH (CSH/Ag) composite. Ag⁺ ions were first embedded into the surface of CSH through ion-exchange and subsequently reduced to Ag NPs by a reducing agent. Due to the high specific surface area and great ion-exchange capacity of CSH, a series of CSH/Ag composites with different silver contents were easily synthesized. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), elemental mapping, X-ray diffraction (XRD), nitrogen sorption/desorption analysis, and inductively coupled plasma-atomic emission spectrometry (ICP-AES) were employed to characterize the synthesized CSH/Ag composites. Moreover, Ag⁺ ions release behaviours from the CSH/Ag composites were investigated and the release profiles showed dose-dependent and long-time sustained release properties. Furthermore, the synthesized CSH/Ag composite displayed good biocompatibility and high antibacterial activities against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Overall, the newly designed CSH/Ag composite is believed to be a promising bactericidal agent in biomedical applications.

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Introduction

In recent years, despite great advancements that have been achieved in inorganic nanomaterial fabrication, generation of well-designed structure and functional composite materials remain an important issue to extend the applications of inorganic nanomaterials. Calcium silicate (CS) materials, owing to their unique properties, including environmentally benign, low cost, good stability, bioactivity, biocompatibility and degradability, have received considerable attention in various fields, such as in drug delivery,^1–3 bone tissue engineering,^4–6 catalyst carrier,^7,8 adsorption and separation.^9–11 In particular, the abundant hydrated Ca²⁺ cations located on the wall can serve as “functional sites” to effectively attach chemical decorations or be used for ion-exchange. Some studies have revealed that drug molecules with acid groups, proteins and metallic ions were rapidly and efficiently adsorbed on calcium silicate hydrate (CSH).^12,13 Moreover, nanoparticles and functional ions have been frequently integrated into CSH to obtain nanocomposites that have displayed improved physicochemical and biological properties.^14–18 This feature makes CSH a powerful host material for the embedded nanoparticles with advanced properties.

As a well-known inorganic nanomaterial, silver nanoparticles (Ag NPs) have gained significant attention because of their excellent bactericidal activity and the wide range of applications in clothing,¹⁹ wound dressing,²⁰ water disinfection,²¹ medical devices,²² and cosmetic products.²³ Morones et al. demonstrated the bactericidal effect of Ag NPs with size ranges from 1 to 100 nm.²⁴ The high surface area of Ag NPs allows more release of Ag⁺ ions to damage the bacterial cell membrane through binding to proteins, deactivating enzymes, and interfering DNA replication.²⁵ Unlike antibiotics, Ag NPs are not yet associated with drug resistance.²⁶ Previous studies have reported that Ag NPs have toxicity towards human cells.^27,28 The toxicity of Ag NPs has been attributed to the uptake of Ag NPs and inhibition of the basic functions in human cells. From this viewpoint, an immobilization of Ag NPs onto an appropriate substrate to inhibit the direct capture of nanoparticles by the cells is an alternative method. Moreover, the immobilized Ag NPs avoid the aggregation and instability of free colloidal Ag and maintain efficient antibacterial ability. Mukherji et al. discovered that the surface immobilized Ag NPs exhibited a greater bactericidal efficiency than that of free colloidal Ag NPs as well as released more Ag⁺ ions into the solution.²⁹ Thus, it is crucial to develop a proper host material to efficiently load Ag NPs.

Nanoscale organic and inorganic materials, such as nanowires, nanotubes, nanosheets and nanoparticles, have been employed as the host substance to achieve Ag NPs immobilization and stabilization.^30–33 Compared to organic substrates, inorganic materials present significant advantages due to their high chemical and thermal stability, versatile surface functionalization, low toxicity, and good biocompatibility. In general, Ag⁺ ions are primarily adsorbed onto the inorganic substance through electrostatic interaction or ion-exchange and are subsequently reduced to Ag NPs by a reducing agent. With a high surface area and specific structure, mesoporous silica,^34,35 zeolite,^36,37 layered double hydroxide,^38,39 halloysite,^40,41 magnetic nanoparticles,^42,43 hydroxyapatite,^44,45 calcium phosphate,^46,47 and silicon nanowires⁴⁸ have been used for immobilizing Ag and these composites showed good antimicrobial activities. Lee et al. found that Ag particles could be easily grown on a calcium phosphate surface and the composite coating showed a controlled release property and high antibacterial activity.⁴⁷ The facile synthesis method was believed to be available for the fabrication of well-designed inorganic antibacterial materials.

In this study, CSH was employed as the host substance to produce a Ag NPs-deposited composite (CSH/Ag) through a facile and effective method. Owing to the high specific surface area and great ion-exchange capacity of CSH, Ag⁺ ions were efficiently embedded into CSH followed by the reduction to Ag NPs. A series of CSH/Ag composites with different silver contents were easily synthesized. Moreover, the Ag⁺ ions release behaviours and the antibacterial activities of the synthesized CSH/Ag composites were investigated. The synthesized CSH/Ag composites are believed to be efficient bactericidal agents for disinfecting the bacteria.

Experimental

Materials and chemicals

Chemical reagents including sodium metasilicate nonahydrate (Na₂SiO₃·9H₂O, 99%), calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O, 99%), and silver nitrate (AgNO₃, 99%) were commercially obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Sodium borohydride (NaBH₄, 98%) was obtained from Aladdin Industrial Corporation (Shanghai, China). Mouse L-929 fibroblast cells were received from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). All the purchased chemicals were of analytical grade and used as received without further purification.

Synthesis of Ag-deposited CSH composite

First, CSH was synthesized by a solution precipitation method. Briefly, 5 mL of aqueous solution containing 708 mg of Ca(NO₃)₂·4H₂O was slowly injected into a 50 mL of aqueous solution containing 852 mg of Na₂SiO₃·9H₂O with magnetic stirring at room temperature. The feeding process was accomplished in 2 h, and then stirring was performed for another 3 h. After centrifugation and thorough washing with water, the CSH product was obtained. Subsequently, 320 mg of the synthesized CSH was redispersed in 50 mL of deionized water, and then a certain amount of AgNO₃ was added to the solution and stirred for 30 min. After centrifugation and thorough washing with water, the product was redispersed in 60 mL of deionized water. Then, 20 mg of NaBH₄ was added and the mixture was stirred for 20 min; the solid product was centrifuged, washed with deionized water and dried for further use. The weights of AgNO₃ used were 5, 10, 20, 30, and 50 mg and the products were labelled as CSH/Ag-5, CSH/Ag-10, CSH/Ag-20, CSH/Ag-30, and CSH/Ag-50, respectively.

Characterization

Transmission electron microscopy (TEM) images were obtained using a HITACHI H-800 transmission electron microscope (HITACHI, Tokyo, Japan). Scanning electron microscopy (SEM) images were obtained using a S-3400 scanning electron microscope (HITACHI, Tokyo, Japan) in secondary electrons mode and a Magellan 400 field-emission scanning electron microscope (FEI, Hillsboro, USA) in electron backscatter mode. X-ray diffraction (XRD) analysis was carried out using D/MAX 2550 VB/PC advance X-ray powder diffraction instrument (XRD, RIGAKU, Japan). The Brunauer–Emmett–Teller (BET) surface area and pore size distribution were determined at 77 K using a Micromeritics Instrument Corporation TriStar II 3020 (Norcross, GA, USA). The concentrations of Ag⁺ ions were determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES) (Shimadzu Scientific Instruments, Kyoto, Japan). UV-visible spectroscopy was performed on a Shimadzu UV-2550 spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Japan).

In vitro Ag⁺ ions release

The kinetics of Ag⁺ ions release was studied from the CSH/Ag composite. A series of CSH/Ag composites (5 mg) with different silver contents were suspended in 5 mL of phosphate buffered saline (PBS, pH = 7.4). The mixtures were shaken in a shaking incubator at 37 °C at 150 rpm for 20 days. At certain intervals of time (0.5, 1, 2, 4, 6, 9, 12, 16, and 20 days), 1 mL of the supernatant was taken out after centrifugation at 5000 rpm for 3 min. The concentrations of Ag⁺ ions were determined by ICP-AES. Each experiment was independently performed three times.

Antibacterial activity test

Escherichia coli (E. coli, ATCC 25922) and Staphylococcus aureus (S. aureus, ATCC 25923) were selected as model Gram-negative and Gram-positive bacteria, respectively. The antibacterial activity of the as-prepared CSH/Ag composite was studied by bacterial kinetic determination. E. coli and S. aureus were cultured in liquid nutrient broth at 37 °C for 12 h, and then the bacterial solution was diluted with liquid nutrient broth until the optical density (OD₆₀₀) of about 0.05 was achieved. Each testing sample was added to a 4 mL of the bacterial solution, and then shaken in a shaking incubator at 37 °C at 150 rpm for 24 h. The growth rates were determined by measuring the OD₆₀₀ values every 4 h. The liquid nutrient broth containing only the bacteria was used as a control group. In addition, the plate count method was also employed to test the antibacterial activity. 100 μL of each bacterial solution was extracted from the mixture after incubation for 24 h, and then diluted and uniformly spread on a solid nutrient agar plate. Following incubation for another 24 h at 37 °C, the plate was counted for viable bacteria. Each experiment was performed in triplicate.

In vitro cell viability evaluation

The cytocompatibility of the prepared materials was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. L-929 fibroblast cells were first seeded at a density of 1 × 10⁴ cells per mL in 96-well plates and incubated for 24 h at 37 °C in a humidified atmosphere with 5.0% CO₂. Then, the sterilized samples were separately added into the wells at concentrations ranging from 10 to 70 mg L⁻¹. After incubation for another 48 h, the cell viability was measured. Each experiment was performed in triplicate.

Results and discussion

Characterization of Ag deposited CSH composite

The procedure for the synthesis of Ag NPs-deposited calcium silicate hydrate (CSH/Ag) composite involved two steps. First, a facile solution precipitation method was adopted for the synthesis of CSH. Then, Ag⁺ ions were embedded into the surface of CSH via ion-exchange, and subsequently reduced to Ag NPs to form the CSH/Ag composite. The morphologies of the prepared products were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Representative SEM images of CSH are shown in Fig. 1a and b. The CSH comprised a hierarchical morphology constructed by the self-assembly of the curved nanosheets, which display mesoporous and macroporous pores. After depositing Ag NPs, the hierarchical structure slightly changed in the CSH/Ag composite. As a representative composite, CSH/Ag-30 (Fig. 1c and d) shows that the Ag NPs are not obvious; this may due to the small size of the immobilized Ag NPs. The SEM photo in the electron backscatter mode and energy dispersive X-ray spectroscopy (EDS) were adopted to determine the Ag NPs and chemical composition of the CSH/Ag-30 composite. As shown in Fig. 2a, abundant and highly disperse Ag NPs with particle sizes about 12 nm existed on the surface of CSH, showing the successful immobilization of Ag NPs. Fig. 2b shows the EDS spectrum of the CSH/Ag-30 composite, and the element peaks of Ca, Si, O, and Ag are found, revealing the basic chemical composition of the CSH/Ag composite.

Fig. 1 SEM images of the synthesized (a and b) CSH and (c and d) representative CSH/Ag-30 composite.

Fig. 2 (a) SEM image in EBS mode and (b) EDS spectrum of the synthesized CSH/Ag-30 composite.

Fig. 3 shows the TEM images of the synthesized CSH and CSH/Ag composites. In comparison to the TEM image of CSH (Fig. 3a), a small quantity of Ag NPs with small size was observed in the CSH/Ag-5 and CSH/Ag-10 composites. However, a large amount of Ag NPs with larger sizes existed in the CSH/Ag-20, CSH/Ag-30 and CSH/Ag-50 composites. In addition, the particle sizes of the immobilized Ag NPs were all below 20 nm in the as-synthesized CSH/Ag composites. Elemental mapping was further employed to investigate the elemental distribution of the representative CSH/Ag composite (CSH/Ag-30). As observed in Fig. 4, Ca, Si, O, and Ag elements were uniformly distributed on the composite, showing the chemical composition and well dispersed Ag NPs.

Fig. 3 TEM images of the synthesized (a) CSH and CSH/Ag composites, (b) CSH/Ag-5, (c) CSH/Ag-10, (d) CSH/Ag-20, (e) CSH/Ag-30, and (f) CSH/Ag-50.

Fig. 4 The EDS elemental mapping for (a) Ca, (b) Si, (c) O and (d) Ag elements from the synthesized CSH/Ag-30 composite.

Furthermore, Fig. 5 shows the digital photos of the powdered products obtained using different concentrations of silver nitrate solution. The initial CSH powder is white, whereas the products (CSH/Ag-5, CSH/Ag-10, CSH/Ag-20, CSH/Ag-30 and CSH/Ag-50) become yellow or brownish after deposition with Ag NPs. The higher the concentrations of silver nitrate used in the preparation procedure, the deeper the colors exhibited by the products.

Fig. 5 Images of the synthesized (a) CSH and CSH/Ag composites powder with different silver content, (b) CSH/Ag-5, (c) CSH/Ag-10, (d) CSH/Ag-20, (e) CSH/Ag-30, and (f) CSH/Ag-50.

The silver content of the as-synthesized CSH/Ag composites were determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES). As shown in Fig. 6, the silver content of the CSH/Ag composites increased with an increase in the initial concentration of AgNO₃. Owing to the high specific surface area and great ion-exchange capacity from the hydrated Ca²⁺ cation of CSH, high content of Ag⁺ ions embedded materials could be firstly obtained. After reduction with the reducing agent, Ag NPs-deposited CSH composites with different silver contents could be easily obtained. Note that the deposited Ag NPs were highly dispersed and the particle sizes were less than 20 nm. This result reveals that the method proposed in this study is suitable for the fabrication of Ag NPs-deposited inorganic materials with tunable silver contents.

Fig. 6 Silver content in the synthesized CSH/Ag composites prepared using different initial AgNO₃ concentrations.

The crystal phase of CSH and CSH/Ag composites were characterized by XRD. As shown in Fig. 7a, the low-intensity diffraction peak at 2θ = 29.4° indicates the poor crystallinity of CSH. However, after ion-exchange with Ag⁺ ions and reduction to Ag NPs, the situation has significantly changed. The characteristic diffraction peaks of Ag NPs (2θ = 38.0°, 44.1°, 64.4° and 77.3°) appear, indicating Ag NPs were well immobilized in the composites. In addition, the relative peak intensity and peak area of Ag NPs in CSH/Ag-50 (Fig. 7f) were higher than those of CSH/Ag-30 (Fig. 7e), CSH/Ag-20 (Fig. 7d), CSH/Ag-10 (Fig. 7c), and CSH/Ag-5 (Fig. 7b) composites, showing higher silver loading amounts in the CSH/Ag-50 composite than in the other four samples.

Fig. 7 XRD patterns of the synthesized (a) CSH and CSH/Ag composites: (b) CSH/Ag-5, (c) CSH/Ag-10, (d) CSH/Ag-20, (e) CSH/Ag-30 and (f) CSH/Ag-50. ●and * indicate the diffraction peaks of CSH and Ag NPs, respectively.

Fig. 8 shows the nitrogen adsorption–desorption isotherms and pore size distribution curves of the as-synthesized CSH and CSH/Ag composites. The Brunauer–Emmett–Teller (BET) specific surface area and BJH desorption cumulative pore volumes are 170.39 m² g⁻¹ and 1.42 cm³ g⁻¹, respectively. The BJH desorption pore size curve of the CSH displays two main pore sizes of 23.2 and 65.0 nm. The high BET specific surface area of CSH may be attributed to the hierarchical structure constructed from the self-assembly of nanosheets. The high specific surface area as well as abundant hydrated Ca²⁺ cations located on the surface make the CSH a promising host substance for the immobilization of metallic ions and metallic nanoparticles. After deposition with Ag NPs, the BET specific surface area values decreased to 162.44, 155.84, 154.73, 151.48, and 137.09 m² g⁻¹ for CSH/Ag-5, CSH/Ag-10, CSH/Ag-20, CSH/Ag-30 and CSH/Ag-50 composites, respectively. The relatively large specific surface still provides a suitable platform for the release of bactericidal agents for the disinfection of bacteria.

Fig. 8 (a and b) Nitrogen adsorption–desorption isotherms and (c and d) pore distribution curves of the synthesized CSH and CSH/Ag composites.

Compared to other inorganic matrix-based Ag antimicrobial materials, CSH matrix possesses several unique characteristics: the abundant hydrated Ca²⁺ cations on the surface is beneficial for the direct immobilization of high amounts of Ag⁺ ions. In contrast, it is difficult to achieve the immobilization for magnetic nanoparticles, silica particles, mesoporous silica and silicon nanowires. On the other hand, the unique hierarchical structure constructed from the self-assembly of curved nanosheets, high specific surface, mesoporous and macroporous pores are especially important for the immobilization of highly dispersed Ag nanoparticles with small sizes, and the features are partially similar to those of Ag NPs-immobilized graphene oxide sheet.⁴⁹

Generally, the antibacterial activity of Ag NPs-based bactericidal agents are usually ascribed to the release of Ag⁺ ions. To evaluate the release of Ag⁺ ions from CSH/Ag composites, the release behaviours were monitored in PBS solution. As shown in Fig. 9, it is observed that Ag⁺ ions rapidly released from the CSH/Ag on the first day and then a slow sustained release is observed over the following 19 days. The cumulative Ag⁺ ions release strongly depends on the silver content and the incubation time. In particular, CSH/Ag composites that had a higher Ag content showed more release. Besides, the sustained Ag⁺ ions release indicated that the CSH/Ag were suitable for a long-term antimicrobial application for the disinfection of bacteria.

Fig. 9 In vitro release profiles of Ag⁺ ions from the CSH/Ag composites: (a) CSH/Ag-5, (b) CSH/Ag-10, (c) CSH/Ag-20, (d) CSH/Ag-30 and (e) CSH/Ag-50.

The in vitro antibacterial activity of the CSH/Ag composite was compared with initial CSH as assessed by the optical density (OD) measurement at 600 nm. Fig. 10a and b illustrate the bacterial growth kinetics of the initial CSH and the CSH/Ag composite against E. coli and S. aureus in liquid nutrient broth for 24 h. When the sample of CSH was added to the E. coli and S. aureus bacteria solutions, no obvious inhibition of the bacterial growth was observed, showing no effective antibacterial activity of CSH. However, compared to the growth kinetics of the control bacteria, the growth kinetics of both E. coli and S. aureus were clearly inhibited by using the CSH/Ag composite. The growth kinetics revealed a typical dose-dependent antimicrobial effect of the CSH/Ag composite. Even on incubation with the CSH/Ag composite with low silver content (e.g., CSH/Ag-5 and CSH/Ag-10), bacteria proliferation was critically retarded. In addition, the higher the Ag content in the CSH/Ag composite, the better the antibacterial effects. The bacterial growth of both E. coli and S. aureus were completely inhibited by the CSH/Ag-30 and CSH/Ag-50 composites during the whole process.

Fig. 10 Bacterial growth kinetics of (a) E. coli and (b) S. aureus treated with CSH and CSH/Ag composites for 24 h.

The bactericidal activities of the CSH/Ag composites were further evidenced by agar plate tests. After 24 h of treatment, the bacterial suspensions were extracted, diluted, and uniformly spread on the solid nutrient agar plates. After incubation at 37 °C for another 24 h, the bacterial colonies were observed. As shown in Fig. 11, the colony forming units were decreased with an increase in the silver content in the CSH/Ag composites. Besides, the images of the agar plate ensured full inhibition of the CSH/Ag-30 and CSH/Ag-50 composite towards both E. coli and S. aureus. Thus, the as-synthesized CSH/Ag composites are highly efficient inhibitors of bacterial growth, and the enhanced antibacterial properties can be achieved by an increase in the silver loading amount.

Fig. 11 Typical images of the re-cultivated bacteria colonies for E. coli and S. aureus after incubation with CSH and CSH/Ag composites.

The in vitro cytocompatibility of the synthesized CSH and CSH/Ag composites were investigated through a cell viability evaluation using L-929 fibroblast cells, and the results are shown in Fig. 12. The viabilities of L-929 cells incubated with CSH were higher than 95%, showing excellent biocompatibility. In case of CSH/Ag composite, low cytotoxicity (cell viability above 80%) was observed in the CSH/Ag groups at a concentration of 10–70 μg mL⁻¹. The results exhibited that the CSH/Ag composites are promising bactericidal agents with good biocompatibility.

Fig. 12 In vitro cytocompatibility assay of CSH and CSH/Ag composites against L-929 cells.

Conclusions

In this study, a facile and efficient method was proposed to produce a silver nanoparticles deposited calcium silicate hydrate composite. The immobilization weight and particle size of silver nanoparticles can be controlled, and a series of composites with highly dispersed and high content of silver nanoparticles were easily obtained. The synthesized CSH/Ag composite exhibited good biocompatibility and strong bactericidal activities towards both Gram-negative and Gram-positive bacteria. It is believed that the CSH/Ag composite has great potential as an efficient bactericidal agent for the disinfection of bacteria in clinical applications.

Notes and references

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