Vancomycin-modified Fe₃O₄@SiO₂@Ag microflowers as effective antimicrobial agents

Introduction

The increasing risk of antibiotic-resistant bacterial infections is a serious problem to health care; this condition decreases the efficiency of conventional antibiotics and significantly increases the number of clinical infections.^1,2 In this regard, scholars have focused on developing new generations of effective antibacterial agents, such as antibacterial nanomaterials, to meet the urgent need of reducing antibiotic abuse.³ The use of nanomaterials, which exhibit high specific surface area and unique physical, chemical, and biological properties, offers new approaches for rapid and effective killing of pathogenic microorganisms.⁴ In contrast to the mechanistic action of antibiotics, nanomaterials target multiple bacterial components; as such, microbes cannot easily acquire resistance toward these particles.⁵ Many nanomaterials, including gold,⁶ silver,⁷ copper,⁸ zinc oxide,⁹ and graphene oxide,¹⁰ exhibit excellent bactericidal activity; of these, Ag nanomaterials possess the optimal antibacterial activity because they kill most pathogenic microbes, including bacteria, fungi, and viruses.^11,12 Thus far, no organism has readily developed resistance to Ag nanomaterials.¹³ Moreover, Ag nanomaterials feature low toxicity to human body and are widely applied in water purification systems, wound dressings, surgery instruments, and implantable materials.¹⁴ Thus, various Ag-based nanoparticles have been extensively studied as potent and broad-spectrum antimicrobial agents.

However, the practical application of these Ag-based nanomaterials is hindered by three limitations that should be addressed urgently. First, the irregular aggregation of small Ag nanoparticles (Ag NPs) reduces or diminishes typical properties associated with their nanoscale size.¹⁵ Ag NPs elicit antibacterial activity due to their nanometer size; that is, small particles exhibit increased activities based on the equivalent Ag mass content.¹⁶ Nevertheless, Ag NPs with a diameter less than 20 nm tend to aggregate, thereby reducing their antibacterial activity.¹⁷ The second issue is the recovery of disinfectants in water environment. The Ag NPs are very small, which makes it difficult to eliminate them completely by centrifugation or filtration, and high concentrations of residual nanosilver in solution can cause potential adverse health effects.¹⁸ Third, Ag-based nanomaterials exhibit inferior activity against Gram-positive bacterial pathogens compared with that against Gram-negative bacteria; this phenomenon could be attributed to the thicker and more stable membrane of the former than that of the latter. This feature prevents the Ag ions from entering into the bacterium.^19,20 However, bacterial infections caused by Gram-positive pathogens are the leading causes of morbidity and mortality in humans.²¹ To overcome these drawbacks, researchers must investigate the combination of Ag NPs with other materials in the field of sterilization.

Recyclability can be achieved using magnetic materials to load Ag NPs, allowing the particles to be easily separated from water solution by magnetic forces.²² Magnetic particles serve as a proper supporting platform for immobilizing Ag particles to form homogeneous ultrafine Ag nanostructures without aggregation.²³ The large surface area of the magnetic nanocomposites allows high loading and sufficient contact area to achieve high antibacterial efficacy. As such, Ag-loaded magnetic particles have been increasingly applied in antibacterial research. Many kinds of Ag-based magnetic composites have been successfully synthesized.^17,24–26 However, the antibacterial effect of the fabricated magnetic composites on Gram-positive bacteria remains insufficient.

On the other hand, a number of researchers reported the synergistic antibacterial effects of metal NPs with conventional antibiotics.^27–30 For example, Brown et al indicated the effective synergistic effect of noble metal NPs functionalized with ampicillin against multiresistant strains.³¹ Lai et al reported a general route for preparing vancomycin-modified Au NPs; the products act as effective broad-spectrum bactericides against both Gram-negative and Gram-positive pathogens.³² These studies have focused on synthesis of different antibiotic-functional nanostructures and investigated their enhanced antibacterial activity. However, the combination of Ag-based magnetic composites and antibiotics as effective antibacterial agents has been rarely reported yet.

In the present study, we propose the use of a novel vancomycin-modified Fe₃O₄@SiO₂@Ag microflower for rapid and effective killing of pathogenic bacteria in solutions. The hybrid microcomposite combines the unique properties of the magnetic core, flower-like Ag shell, and modified-vancomycin layer. The microcomposite shows significantly enhanced antibacterial activity and is environment friendly. High-performance Fe₃O₄@SiO₂@Ag microflowers with good dispersity, uniform structure, and sufficient magnetic responsiveness were prepared via our proposed sonochemical-based method and used as the core component.³³ Compared with the conventional smooth Ag shell, the highly branched flower-like Ag shell provided larger surface area for effective Ag ion release and bacterial contact. Vancomycin was selected for binding to the Fe₃O₄@SiO₂@Ag microflowers to maximize the bactericidal potential; this antibiotic exhibits a well-established mechanism and synergistic antibacterial effects with various types of NPs.^34,35 Vancomycin can directly bind to the terminal D-Ala-D-Ala portion of the peptidoglycan of the cell wall of a Gram-positive bacterium through hydrogen bonds to inhibit peptidoglycan synthesis. Moreover, previous studies have demonstrated that vancomycin-modified NPs elicit enhanced antimicrobial activities or can electively capture not only Gram-positive but also Gram-negative bacterium.^36,37 The fabricated Van/Fe₃O₄@SiO₂@Ag microflowers were subjected to antibacterial tests against both Gram-positive and Gram-negative bacteria, and we concluded that a combination of vancomycin and Fe₃O₄@SiO₂@Ag microflowers as antibacterial agents could lead to better antimicrobial activity through their synergistic effects. To the best of our knowledge, this study is the first to investigate the synergistic effect of magnetic-based Ag microcomposites and vancomycin for sterilization. Additionally, the recyclability of Van/Fe₃O₄@SiO₂@Ag microflowers was evaluated over five washing-and-reuse cycles to determine their potential in practical applications.

Experimental methods

Materials and chemicals

Tetraethoxysilane (TEOS), n-butylamine, silver nitrate, vancomycin, polyvinylpyrrolidone (PVP, 40 kDa), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), 11-mercaptoundecanoic acid (MUA), and 2-(N-morpholino)ethanesulfonic acid (MES) were obtained from Sigma-Aldrich Chemicals Co. (St Louis, MO, USA). Ferric chloride (FeCl₃·6H₂O), ethylene glycol (EG), diethylene glycol (DEG), trisodium citrate, sodium acetate anhydrous (NaOAc), and phosphate-buffered saline (PBS) buffer (10 mM, pH 7.4) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, People’s Republic of China). All chemicals were of analytical grade and were utilized as received unless mentioned otherwise. All aqueous solutions were purified with a Milli-Q system (18.2 MO cm⁻¹). Commercial Escherichia coli (BL21) and clinical isolation strains of methicillin-resistant Staphylococcus aureus (MRSA) were provided by the Institute for Disease Control and Prevention, Academy of Military Medical Sciences.

Synthesis of Fe₃O₄ and Fe₃O₄@SiO₂ microspheres

Superparamagnetic Fe₃O₄ particles (∼200 nm) were synthesized by modified solvothermal reaction.³⁸ In a typical procedure, 1 g of FeCl₃·6H₂O (≈4 mmol) was dissolved in a mixture of DEG and EG (V_DEG/V_EG =13/7; total volume of 40 mL) in an explosion-proof bottle under magnetic stirring. The solution was added with 4 g of PVP and heated at 120°C for 30 min until a transparent solution was obtained. Then, the solution was added with 3 g of NaOAc, and heating was terminated. After stirring for an additional hour, the obtained homogeneous solution was sealed in a Teflon-lined stainless steel autoclave (100 mL volume) and then transferred to a vacuum oven, which was preheated to 200°C. After a 10-h reaction period, the solution was cooled to room temperature, and the obtained Fe₃O₄ particles were collected with an external magnet, washed five times with ethanol and water, and then dried in vacuum at 60°C for 6 h.

The synthesis of Fe₃O₄@SiO₂ microspheres was carried out according to a modified Stöber method. Briefly, 0.1 g of as-prepared Fe₃O₄ microspheres was dispersed in a mixture of ethanol–water–ammonia (180 mL/10 mL/8 mL) by sonication for 15 min. Subsequently, 0.3 mL of TEOS solution was consecutively added to the mixture under vigorous sonication for 1 h. The resulting Fe₃O₄@SiO₂ microspheres were magnetically separated and washed and then redispersed in 10 mL of anhydrous ethanol.

Synthesis of Fe₃O₄@SiO₂–Ag seed microspheres

To synthesize Fe₃O₄@SiO₂–Ag seed microspheres, 0.1 g of Fe₃O₄@SiO₂ microspheres was thoroughly washed with anhydrous ethanol and then dispersed in 1 mL of anhydrous ethanol by ultrasonic irradiation. In a typical deposition procedure, the cleaned Fe₃O₄@SiO₂ microspheres were mixed with 9 mL of the silver nitrate solution (0.8 mM) and incubated at 30°C for 30 min under sonication. Then, the reaction tube was then added with 80 μL of butylamine (100 mM). The mixture was ultrasonically irradiated for 1 h to allow the deposition of small and dense Ag seeds onto the Fe₃O₄@SiO₂ surface. Finally, the products were magnetically separated from the suspension and redispersed in ethanol, and the solution was called Fe₃O₄@SiO₂–Ag seed solution.

Synthesis of Fe₃O₄@SiO₂@Ag microflowers

Briefly, 1 mL of the prepared Fe₃O₄@SiO₂–Ag seed was added into 400 mL of the AgNO₃ aqueous solution (0.2 mM) to obtain high-performance Fe₃O₄@SiO₂@Ag microflowers. The mixture was added with 400 μL of 37% formaldehyde and 800 μL of 25% ammonia solution successively. After a 5-min reaction period, the solution was added with 600 mg of PVP powder and sonicated for another 15 min. Finally, the products were collected and rinsed several times with deionized water to remove excess PVP.

Immobilization of vancomycin onto the surface of Fe₃O₄@SiO₂@Ag microflowers

For the preparation of vancomycin-modified Fe₃O₄@SiO₂@Ag microflowers, the microflowers were carboxyl-group-functionalized first and then conjugated with vancomycin according to a previously reported method.³⁶ In brief, Fe₃O₄@SiO₂@Ag microflowers (10 mg/mL) were incubated overnight in an MUA ethanol solution (20 μM) to obtain surface-carboxylated microflowers. Then, the prepared MUA/Fe₃O₄@SiO₂@Ag microflowers in 10 mL of MES buffer (0.1 M, pH 5) were mixed with 1 mL of EDC (10 mg/mL) and sonicated for 5 min. The mixture was then added with 1 mL of vancomycin (5 mg/mL) and sonicated for 2 h. The resulting Van/Fe₃O₄@SiO₂@Ag microflowers were washed with water and redispersed in PBS buffer (10 mM, pH 7.4).

Material characterization

Scanning electron microscopy (SEM) images were taken on a JEOL JSM-7001F microscope operating at 10 kV. Transmission electron microscopy (TEM) images were taken with a Hitachi H-7650 microscope operating at 80 kV. High-resolution TEM (HRTEM) images and energy-dispersive X-ray spectroscopy (EDX) spectra were obtained using a JEOL JEM-2010F microscope at an accelerating voltage of 200 kV. UV–vis spectra were obtained using a Shimadzu 2600 spectrometer. Magnetic characterization was conducted with a superconducting quantum interference device magnetometer (SQUID, MPMSXL-7) at 300 K. The X-ray diffraction (XRD) patterns of the products were investigated by a Japan Rigaku D/max 2550 VB/PC rotation anode X-ray diffractometer. Zeta potential of products was measured by dynamic light scattering with Zetasizer Nano ZS (NANO-ZS 90; Malvern Instruments, Malvern, UK). A simultaneous differential scanning calorimetry/thermogravimetric analysis (DSC/TGA) was performed to characterize the decomposition and thermal stability of products measured from 30°C to 600°C at the heating rate of 10°C/min.

Antibacterial tests

For the antibacterial activity tests, two bacterial species, Gram-negative E. coli BL21 and Gram-positive MRSA, were used in the experiments. E. coli BL21 was cultured in Luria–Bertani medium, and MRSA was cultured in trypticase soy broth medium. After 8 h, the concentration of the bacteria was determined by the optical density (OD) at 600 nm based on the turbidity of the cell suspension.³⁹ Subsequently, the bacterial suspension was diluted by PBS buffer (10 mM, pH 7.4), and a final density of 10⁴ cells/mL was obtained for the antibacterial tests. One hundred microliters of the bacterial suspension was inoculated in 10 mL of the culture medium supplemented with different amounts of Van/Fe₃O₄@SiO₂@Ag microflowers. The suspensions were shaken at 200 rpm at 37°C for 10 h, and then the bacterial survival was withdrawn and determined by measuring the absorbance at 600 nm. The control group was prepared under the similar condition in the absence of Van/Fe₃O₄@SiO₂@Ag microflowers and the presence of Fe₃O₄@SiO₂@Ag microflowers for comparison. Classical culture method was performed to study the antibacterial activity of Fe₃O₄@SiO₂@Ag microflowers and vancomycin-modified microflowers. One hundred microliters of the above-treated bacterial suspension was diluted by 10⁵ times serially by PBS buffer and directly cultured on an agar plate and incubated at 37°C. After 24 h, the number of colonies was counted to evaluate the survival condition of the bacterial cells.

Results and discussion

Fabrication of vancomycin-modified Fe₃O₄@SiO₂@Ag microflowers

As illustrated in Scheme 1A, vancomycin-modified Fe₃O₄@SiO₂@Ag microflowers were fabricated in five steps. Superparamagnetic Fe₃O₄ particles (~200 nm) used as the magnetic core were synthesized through a modified solvothermal reaction at 200°C. Then, Fe₃O₄@SiO₂ microspheres were prepared by coating the Fe₃O₄ core with a thicker silica shell (~50 nm) via a fast sol–gel method using sonication. The silica shell of magnetic structure efficiently dispersed the particles in the aqueous solution and provided functional groups for deposition of the Ag seeds. We employed a butylamine reduction system to deposit dense Ag seeds onto the surface of the Fe₃O₄@SiO₂ particles. Subsequently, the Fe₃O₄@SiO₂@Ag microflowers were synthesized through ultrasound-assisted seed-mediated growth strategy according to our previous publication.³³ All Ag seeds isotropically grew within a few seconds and were stabilized by PVP. Finally, vancomycin-coated microflowers were synthesized via a two-step coupling method: 1) Fe₃O₄@SiO₂@Ag microflowers were carboxyl-group-functionalized through MUA modification and 2) the vancomycin was modified on the surface of the carboxylated Fe₃O₄@SiO₂@Ag microflowers through carbodiimide chemistry.

Scheme 1 (A) Synthesis route of Van/Fe3O4@SiO2@Ag microflowers. (B) Schematic of the operating procedures for bacterial killing in solution by Van/Fe3O4@SiO2@Ag microflowers. Abbreviation: TEOS, tetraethoxysilane.

Characterization of Fe₃O₄@SiO₂@Ag microflowers

TEM and SEM analyses were applied to characterize the morphology of the as-obtained products during different stages. Figure 1A shows the typical TEM image of the monodispersed Fe₃O₄ particles with a diameter of ~200 nm. The uniform Fe₃O₄ spherical particles were synthesized by the modified solvothermal method, where PVP was used to improve the water dispersibility. The silica coating was created by the simple sol–gel process. The silica shell thickness could be easy controlled by varying the concentration of TEOS.⁴⁰ Figure 1B presents the TEM image of uniform Fe₃O₄@SiO₂ microspheres with an obvious core/shell structure. The silica shell is clearly visible and possesses ~50 nm thickness. The zeta potential of Fe₃O₄@SiO₂ in ethanol was −31.3 mV, indicating that the surface hydroxyl groups of silica shell were deprotonated in absolute ethanol (Figure S1). The negatively charged Fe₃O₄@SiO₂ particles adsorbed Ag⁺ ions as nucleation centers for further Ag seed formation. After the Ag⁺ ions were bound to the SiO₂ shell sufficiently, AgNO₃ was reduced by butylamine and conjugated to the silica surface. Figure 1C shows that the dense and relatively uniform Ag seeds successfully coated the Fe₃O₄@SiO₂ particles. The size of the deposited seeds was ~10–20 nm. The deposition of the Ag seeds was also confirmed based on the SEM image (Figure 1E), which shows that these particles are well distributed on the Fe₃O₄@SiO₂ microspheres. Butylamine is a mild reductant; as such, excess nucleation centers were not formed in the solution.⁴¹ The particle size and density of the Ag seeds on the SiO₂ surface can be controlled by tuning the concentrations of butylamine and AgNO₃.⁴² Figure S2 shows the Fe₃O₄@SiO₂–Ag seed particles synthesized with different conditions. The TEM images of all the seed particles were obtained after the deposition of the Ag particles in a mixture of AgNO₃ and butylamine at a molar ratio of 1:1. When 0.2, 0.4, and 0.8 mM AgNO₃ and butylamine were added to the Fe₃O₄@SiO₂ ethanol solution, abundant and small Ag seeds were deposited uniformly on the SiO₂ surface. Fe₃O₄@SiO₂–Ag seed synthesized with 0.8 mM AgNO₃ and butylamine was found to be the optimal seed particle for flower-like Ag petal growth. Seed-mediated growth was determined as the mechanism underlying the formation of the flower-like Ag shell produced by a one-step redox reaction in the presence of suitable Ag seeds deposited on the Fe₃O₄@SiO₂ microspheres. The flower-like shell formation was rapidly completed by ultrasound-driven synthesis, where the Ag seeds acted as nucleation sites for rapid anisotropy. Superfluous AgNO₃ is necessary in the reaction system because a high reactant concentration promotes the formation of highly branched structures. The fast-growing Ag petals consumed large amounts of Ag atoms. When ammonia was injected, Ag⁺ ions were reduced by CH₂O immediately and quickly deposited on the Ag seeds of silica surface, resulting in the formation of flower-like Ag shells surrounding the magnetic cores. Figure 1D shows the TEM picture of the obtained Fe₃O₄@SiO₂@Ag microflowers, which possess a highly branched morphology and are nearly uniform with a particle diameter ranging from 700 to 900 nm. The SEM image in Figure 1F shows that the microflowers are composed of numerous flower-like Ag petals and ultrasharp Ag tips. All the magnetic-based Ag microflowers exhibit relatively uniform morphology and size. Furthermore, the TEM and SEM images indicate the successful controllable synthesis of high-performance Fe₃O₄@SiO₂@Ag microflowers. PVP was used as a stabilizer to limit particle aggregation. In sample without PVP, Fe₃O₄@SiO₂@Ag microflowers were formed but would be easily aggregated (Figure S3A); this aggregation process is irreversible. When the concentration of PVP reached 1 mg/mL, the dispersibility of the microflower particles improved (Figure S3B).

Figure 1 Characterization of Fe3O4@SiO2@Ag microflowers. Transmission electron microscopy images of (A) Fe3O4, (B) Fe3O4@SiO2, (C) Fe3O4@SiO2–Ag seed, and (D) Fe3O4@SiO2@Ag microflowers. Scanning electron microscopy images of (E) Fe3O4@SiO2–Ag seed and (F) Fe3O4@SiO2@Ag microflowers. (G) X-ray diffraction patterns and (H) energy-dispersive X-ray spectra of Fe3O4@SiO2@Ag microflowers. The circles represent the peaks of the magnetite structure of Fe3O4. The squares indicate the peaks of cubic Ag.

The as-prepared samples were also examined by XRD analysis (Figure 1G). Curve a in Figure 1G shows the XRD spectra of the Fe₃O₄@SiO₂ microspheres, which fitted well to the Fe₃O₄ bulk phases (JCPDS card no 75-1609).^43,44 As the SiO₂ shell was amorphous, no diffraction peaks corresponding to SiO₂ were observed. With the deposition of Ag seeds, four additional diffraction peaks were observed at 2θ values of 38.2°, 44.3°, 64.5°, and 77.5°, which correspond to the reflections of the (111), (200), (220), and (311) crystal planes of Ag, respectively (curve b of Figure 1G). These peaks correspond to the cubic-phase Ag (JCPDS no 04-0783) and indicate that dense Ag seeds were successfully deposited on the surface of Fe₃O₄@SiO₂ microspheres.^45,46 The XRD pattern of the Fe₃O₄@SiO₂@Ag microflowers showed an obvious increase in the intensity of the Ag peaks when the highly branched Ag shell was formed outside the magnetic core (curve c of Figure 1G). The EDX spectrum (Figure 1H) of the Fe₃O₄@SiO₂@Ag microflower confirms that the microflower particles mainly consist of Fe, Si, O, and Ag. No other impurity peaks were detected in the EDX spectrum, indicating the purity of the final product.

The UV–vis absorbance of the as-obtained products is shown in Figure 2A. Curves a and b denote the typical UV–vis spectra of Fe₃O₄ and Fe₃O₄@SiO₂ microspheres, respectively. After the small Ag seeds were deposited, a broad absorption peak appeared at ~450 nm due to the Mie plasmonic resonance from the neighboring silver particles (curve c of Figure 2A).⁴⁷ After the flower-like shell was formed outside the magnetic core, the obvious absorption peak became strong and blue-shifted to ~375 nm (curve d of Figure 2A). This phenomenon can be ascribed to the strong plasmon coupling between neighboring Ag petals. Hence, the highly branched Ag shell coated the magnetic core.

Figure 2 (A) UV–vis spectra and (B) magnetic hysteresis curves of the prepared Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2–Ag seed microspheres, and Fe3O4@SiO2@Ag microflowers. Insets in (A) and (B) show suspensions before and after magnetic enrichment by an external magnetic force, respectively.

The magnetic properties of as-prepared samples were investigated with a SQUID magnetometer. The saturation magnetization (MS) values of Fe₃O₄, Fe₃O₄@SiO₂, Fe₃O₄@SiO₂–Ag seed, and Fe₃O₄@SiO₂@Ag microflowers were 85.2, 61.7, 53.5, and 21.4 emu g⁻¹, respectively (Figure 2B). The MS values gradually decreased after SiO₂ coating, Ag seed deposition, and flower-like Ag shell formation. This result can be explained by the increasing number of nonmagnetic coating materials formed around the Fe₃O₄ cores. Despite the significant decrease in the MS value of the Fe₃O₄@SiO₂@Ag microflowers, the magnetization of the final structure remained sufficient for fast magnetic separation. All of the curves nearly intersected with the origin, indicating that the synthesized products were in a superparamagnetic state at room temperature. These excellent magnetic properties confer the microflowers with great potential in bioseparation and biodetection.

Characterization of vancomycin-modified Fe₃O₄@SiO₂@Ag microflowers

Figure 3A shows the modification method for depositing vancomycin molecules onto the surface of the fabricated Fe₃O₄@SiO₂@Ag microflowers. MUA was used to form a bifunctional linker, which not only provided terminal carboxyl group for subsequent coupling reaction but also acted as a suitable arm for reducing the steric hindrance between vancomycin and bacteria. MUA-modified Fe₃O₄@SiO₂@Ag microflowers were easily obtained by treating MUA under sonication with the formation of strong covalent Ag–S bond.⁴⁸ The carboxyl groups on the Fe₃O₄@SiO₂@Ag microflowers surface were bound with vancomycin via carbodiimide activation to yield vancomycin-modified Fe₃O₄@SiO₂@Ag microflowers. HRTEM analysis was performed to obtain detailed insights into the surface structure of the Van/Fe₃O₄@SiO₂@Ag microflowers (Figure 3B and C). The HRTEM picture obtained from the edge of flower-like Ag shells (Figure 3C) shows that the Fe₃O₄@SiO₂@Ag microflowers are surrounded by a thin membrane-like layer with ~1.5 nm thickness. This membrane-like structure was formed by MUA and vancomycin. To confirm that vancomycin was successfully modified on the Fe₃O₄@SiO₂@Ag microflowers, we used elemental mapping for analysis of the presence and spatial distribution of the N component. Figure 3D shows the bright-field TEM image of a part of an individual Fe₃O₄@SiO₂@Ag microflower. Figure 3E–I shows the elemental mapping of the sample, where Fe is located in the magnetic core, Si and Ag are distributed throughout the entire composite, and N is mainly located on the surface of the microflower. In the designed Van/microflower structure, N was only derived from the vancomycin molecules. Therefore, the intensive N signal confirms that Fe₃O₄@SiO₂@Ag microflower was successfully conjugated with vancomycin.

Figure 3 Characterization of Van/Fe3O4@SiO2@Ag microflowers. (A) Schematic of the molecular structure of vancomycin and its coupling interaction with Fe3O4@SiO2@Ag microflowers. (B) High-resolution transmission electron microscopy image of the vancomycin-modified Fe3O4@SiO2@Ag microflower and (C) the corresponding magnified image obtained from the circled area in (B). Arrows indicate a thin membrane-like layer of vancomycin and 11-mercaptoundecanoic acid. (D) The bright-field transmission electron microscopy image of a part of an individual Fe3O4@SiO2@Ag microflower. (E–I) The associated elemental mappings showing the element distributions of Fe (red), Si (green), Ag (yellow), and N (blue) in the composite.

The UV–vis absorption spectra of vancomycin solution before and after the conjugation reaction were also examined (Figure S4). The absorption intensity at 280 nm of the supernatant of the MUA/Fe₃O₄@SiO₂@Ag microflowers significantly decreased after vancomycin conjugation. This result further confirms that the vancomycin molecules were effectively bound to the Fe₃O₄@SiO₂@Ag microflowers surface. Moreover, a simple experiment was carried out to investigate the bonding strength of vancomycin molecules to the Fe₃O₄@SiO₂@Ag microflowers. In this test, 0.1 g of the Van/Fe₃O₄@SiO₂@Ag microflowers was continuously stirred in 10 mL of distilled water at room temperature overnight. A filtered sample of the solution was obtained, and its UV–vis spectrum was recorded. Vancomycin showed an absorption band at ~280 nm in its UV–vis spectrum. However, the spectrum of the filtered solution did not show any absorption band in this range. This finding indicates the absence of vancomycin in the filtered solution, suggesting the strong coupling of vancomycin molecule to the Ag microflowers.

Antibacterial properties of vancomycin-modified Fe₃O₄@SiO₂@Ag microflowers

We evaluated the sterilizing effect of the synthesized Van/Fe₃O₄@SiO₂@Ag microflowers on E. coli and MRSA as model Gram-negative and Gram-positive bacteria, respectively, both of which represent the leading multidrug-resistant bacterial pathogens. Bacterial inhibition growth curves were plotted to investigate the antibacterial abilities of the Fe₃O₄@SiO₂@Ag microflowers alone and in combination with vancomycin. Diluted solutions of the products were incubated with 10 mL of bacterial suspension (about 10⁴ CFU/mL), and the control group was subjected to a similar condition in the absence of synthesized microflowers for comparison. The results of bacterial proliferation were determined by the OD at 600 nm of the suspension. The concentration-induced antibacterial results are shown in Figure 4A and B. The fabricated Van/Fe₃O₄@SiO₂@Ag microflowers slowed down the growth of both strains (E. coli and MRSA). Remarkably, the minimum inhibitory concentrations (MICs) of the vancomycin-modified Fe₃O₄@SiO₂@Ag microflowers were 10 μg mL⁻¹ for E. coli and 20 μg mL⁻¹ for MRSA. Moreover, the antibacterial ability of Van/Fe₃O₄@SiO₂@Ag microflowers was significantly higher than that of the nonmodified microflowers. For both strains, Fe₃O₄@SiO₂@Ag microflowers at a concentration of 20 μg mL⁻¹ did not restrain the bacterial growth after 5 h, whereas the Van/Fe₃O₄@SiO₂@Ag microflower-treated group showed almost 100% killing efficiency under the same experimental condition. The Fe₃O₄@SiO₂@Ag microflowers could be an efficient antibacterial agent because its flower-like Ag shell provides larger active surface areas for contacting bacteria and releasing Ag ions than those of conventional smooth Ag shell. Figure S5 shows the result of incubating the same bacterial sample with the Fe₃O₄@SiO₂@Ag microflower alone overnight. The MIC values of Fe₃O₄@SiO₂@Ag microflowers were 25 μg mL⁻¹ for Gram-negative E. coli and 55 μg mL⁻¹ for Gram-positive MRSA. We conclude that the MIC decreased by about 60% for E. coli and 63.5% for MRSA when using only Fe₃O₄@SiO₂@Ag microflowers.

Figure 4 (A and B) Bacterial growth curves in culture media with Van/Fe3O4@SiO2@Ag microflowers. Different concentrations of vancomycin-modified Ag microflowers were used in the cultures of (A) Escherichia coli and (B) methicillin-resistant Staphylococcus aureus (MRSA). The growth of the bacteria was measured by determination of optical density (OD) at wavelength of 600 nm. (C) Photographs showing viability of E. coli and MRSA after treatment under different synthesized products at a concentration of 20 μg mL−1: (i) control group, (ii) Fe3O4@SiO2, (iii) Fe3O4@SiO2–Ag seed, (iv) Fe3O4@SiO2@Ag microflowers, (v) Van/Fe3O4@SiO2@Ag microflowers, and (vi) free-form vancomycin.

Plate counting method was carried out to assess the antibacterial ability of the obtained microstructures in different stages.⁴⁹ Approximately 100 μL of the overnight-cultured bacterial suspension was diluted to 10³ cells/mL with medium, and then spread onto the agar plates, and cultured at 37°C overnight. Figure 4C shows the formation of bacterial colonies in the control and treated groups of different microstructures after 12-h cultivation. The control (absence of particles) and Fe₃O₄@SiO₂ microsphere-treated solutions did not inhibit the bacterial growth. The Fe₃O₄@SiO₂–Ag seed showed higher antibacterial efficiency compared with its precursor Fe₃O₄@SiO₂ microspheres. After the decoration of Ag seeds and flower-like Ag petals, the number of E. coli and MRSA colonies gradually decreased. These results prove that Ag-coated magnetic microcomposites exhibit antibacterial activities against both Gram-negative and Gram-positive bacterial pathogens. The bactericidal effect also increased significantly with increasing Ag coating components surrounding the magnetic core. Few colonies of E. coli were found during treatment with the Fe₃O₄@SiO₂@Ag microflowers, whereas bacterial colonies of MRSA were still observed in Ag microflower-treated group. MRSA showed less susceptibility to the Fe₃O₄@SiO₂@Ag microflowers than E. coli. The Fe₃O₄@SiO₂@Ag microflowers cannot effectively kill MRSA at 20 μg mL⁻¹ within 60 min. However, almost no colonies of both Gram-negative (E. coli) and Gram-positive (MRSA) bacterial strains were observed during treatment with the Van/Fe₃O₄@SiO₂@Ag microflowers, indicating the inactivation of most bacteria. Picture (vi) of Figure 4C shows the photograph taken from the vancomycin-treated bacteria sample that was incubated for 1 h. The colony number was slightly smaller than that observed in the control groups, indicating that vancomycin molecules are not effective at 20 μg mL⁻¹ in such a short time. Hence, the activity of the fabricated Van/Fe₃O₄@SiO₂@Ag microflowers against bacteria, especially MRSA, was enhanced when combined with vancomycin. These results indicate the presence of synergistic effect between the microflowers and vancomycin.

Fluorescence-based cell viability assay was performed to determine the bacterial survival rate and demonstrate the relation between the bactericidal effect and the sterilization duration at a fixed concentration of Van/Fe₃O₄@SiO₂@Ag microflowers (20 μg mL⁻¹). The live/dead staining technique contains two nucleic dyes, SYTO9 and propidium iodide (PI), to quantify live and dead microbes from the fluorescence microscopy images.¹¹ The SYTO9 can enter bacterial cell membrane and label DNA to produce green color for living bacteria, while PI only binds to the nucleic acid of the dead cells with destroyed membrane and shows red fluorescence. As shown in Figure 5A, the number of live bacteria decreased with prolonged sterilization. Almost no red fluorescence can be observed at the beginning of the test, which indicated that most bacteria are alive. After the bacteria were incubated with the Van/Fe₃O₄@SiO₂@Ag microflower solution for different durations, almost all E. coli colonies were killed in less than 45 min, whereas 99% of MRSA colonies were killed within 60 min. Thus, the bactericidal activity of Van/Fe₃O₄@SiO₂@Ag microflowers depends on the concentration of the microflowers and the incubation time. The representative time-dependent bacterial viability rates are shown in Figure 5B. We finally examined the morphological changes in the bacterial cells after the antimicrobial treatment. Figure 5C and D shows the SEM micrographs of E. coli and MRSA after incubation with Van/Fe₃O₄@SiO₂@Ag microflowers for 60 min, respectively. Treated rod-like E. coli cells (Figure 5C) showed extensive membrane damage and leaked intracellular components, resulting in shrinkage and lysis of cells. Similarly, extensive membrane lysis and significant reduction in the size of the treated grape-like MRSA cells were observed (Figure 5D). The significant bacterial cell damage and changes in the cell morphology within such a short time could be mainly due to the interactions of Van/Fe₃O₄@SiO₂@Ag microflowers with the cell wall; this interaction enhanced permeability of the membrane and accelerated bacterial cell disruption. These results further demonstrate the rapid and efficient sterilization capacity of the synthesized Van/Fe₃O₄@SiO₂@Ag microflowers.

Figure 5 (A) Fluorescence microscopy images of live and dead Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) treated with 20 μg mL−1 Van/Fe3O4@SiO2@Ag microflowers for different durations. The red color indicates dead bacteria, and the green color denotes live bacterial cells. Scale bar is 20 μm. (B) Percentage survival of bacterial cells (counted with live/dead bacterial kit). Data are expressed as mean ± standard deviation (n=3). (C and D) Scanning electron microscopy images of E. coli and MRSA after 60 min of incubation with Van/Fe3O4@SiO2@Ag microflowers.

Recyclability of Van/Fe₃O₄@SiO₂@Ag microflowers

Recyclability is the main advantage of the fabricated Van/Fe₃O₄@SiO₂@Ag microflowers over other traditional antibacterial nanomaterials. The magnetic property renders the Van/Fe₃O₄@SiO₂@Ag microflowers to be facilely controlled by an external magnetic force after bactericidal behavior and makes the recovered microcomposites ready for a new antimicrobial cycle (Scheme 1B). Recyclability tests were performed with E. coli (10 μg mL⁻¹) and MRSA (20 μg mL⁻¹) using plate counting method; after five magnetic separation cycles, the antimicrobial effect was maintained at ~95% and 90%, respectively. This result suggests that the recycled Van/Fe₃O₄@SiO₂@Ag microflowers remained stable and exhibited excellent sterilization abilities (Figure 6). The decrease in the antimicrobial effect may be due to leakage of Ag during incubation in bacterial solution or covering of the active vancomycin-modified Ag surface by interferents, such as peptidoglycan, proteins, and nucleic acids, released from the killed bacterial cells. The chemical structure of the Van/Fe₃O₄@SiO₂@Ag microflowers was checked after the recycling by TGA and DSC analysis. No big differences were found between the materials before and after recycling which indicated that the chemical structure was maintained after five antimicrobial cycles (Figure S6). The potential recyclability of the magnetic microflowers provides a novel and green approach toward eliminating the one-time-use problem of traditional antibacterial materials.

Figure 6 Antibacterial effect of recycled Van/Fe3O4@SiO2@Ag microflowers against Escherichia coli (10 μg mL−1) and methicillin-resistant Staphylococcus aureus (20 μg mL−1). The error bars represent the standard deviation of the experiments performed in triplicate.

Antibacterial mechanism of Van/Fe₃O₄@SiO₂@Ag microflowers

Overall, the results of real-time bacterial growth monitoring, agar plate colony analysis, and live/dead cell staining observations indicate the enhanced antibacterial efficiency of the fabricated Van/Fe₃O₄@SiO₂@Ag microflowers against both Gram-negative (E. coli) and Gram-positive (MRSA) bacteria as well as the poor bactericidal effect of the same concentration of Fe₃O₄@SiO₂@Ag microflowers against MRSA. The mechanism underlying the synergistic effect of the Ag microflower and vancomycin must be further clarified. Studies showed that the antimicrobial properties of Ag materials rely on free Ag ions, which can not only bind to sulfur, oxygen, and nitrogen of essential enzymes, resulting in bacterial cell death, but also react with DNA to prevent unwinding, resulting in serious damage to bacterial cells.^29,50,51 Moreover, the Ag NPs itself can directly deactivate the sulfur-containing proteins of cell membrane, causing functional damage to the membrane and the structural changes.⁵²

On the other hand, vancomycin is a potent antibiotic against Gram-positive bacteria and exerts antibacterial activity by forming hydrogen bonds with the peptidoglycan terminus D-Ala-D-Ala on the cell wall. This phenomenon inhibits the synthesis of the bacterial cell wall. Moreover, Gram-negative bacteria can be captured by vancomycin because of either unspecific binding between receptors on the pathogen surface and the glycosides on the vancomycin moiety or deformities/breaks in the outer membrane of Gram-negative bacteria, exposing the D-Ala-D-Ala groups on the interior bacterial surface.³⁷ Therefore, vancomycin-modified nanostructures can interact strongly with a broad range of Gram-positive and Gram-negative bacteria and thus can be used as affinity probes to selectively trap these organisms.^21,53

On the basis of the knowledge in the previous studies combined with our experimental results, the plausible mechanism of the enhanced antibacterial activity of Van/Fe₃O₄@SiO₂@Ag microflowers is proposed in Figure 7A. The Van/Fe₃O₄@SiO₂@Ag microflower can interact with the cell wall of the bacteria and adhere onto the bacterial surface through its surface-modified vancomycin layer. Through direct contact, the vancomycin layer plays important roles in destroying the structure of the cell wall and increasing the permeability of the cell membrane. Moreover, the highly branched petals of the Ag microflowers act as extended arms in highly active surface areas for bacterial contact and Ag ion release. This phenomenon leads to rapid cell wall lysis and enhanced penetration of the Ag ions into the bacterium. The TEM studies clearly show that the Van/Fe₃O₄@SiO₂@Ag microflowers can effectively bind to both E. coli and MRSA, and the Ag petals of the microflowers are closely attached to the cell wall (Figure 7B–G). We believe that vancomycin-modified Ag petals interact with the cell membrane and adhere onto the bacterial surface, leading to disorganized permeability of the cell membrane, speeding up Ag ions entry into bacterium, and eventually causing cell death.

Figure 7 (A) Mechanism of the synergistic antibacterial effect of vancomycin with Fe3O4@SiO2@Ag microflowers. Transmission electron microscopy images of (B) Escherichia coli (control) and (C and D) E. coli treated with Van/Fe3O4@SiO2@Ag microflowers, and (E) methicillin-resistant Staphylococcus aureus (MRSA) (control) and (F and G) MRSA treated with Van/Fe3O4@SiO2@Ag microflowers. (D and G) Increased magnifications of (C) and (F), respectively.

Conclusion

A newly developed Van/Fe₃O₄@SiO₂@Ag microflower with enhanced antibacterial activity is experimentally and theoretically presented in this study. The obtained microcomposite comprised three parts: a submicron-scale magnetic core to provide sufficient magnetic response property, a flower-like Ag shell to build highly active areas for Ag ion release and bacterial contact, and an ultrathin vancomycin layer to bind to the bacterial cell wall and increase the cell membrane permeability. The detailed nanostructures of the products were characterized by HRTEM, SEM, EDX, XRD, and elemental mapping analyses. The Van/Fe₃O₄@SiO₂@Ag microflowers can rapidly and effectively kill both E. coli and MRSA at low concentrations. The killing efficiency can be increased to a high level. The MIC of the enhanced effect of the Van/Fe₃O₄@SiO₂@Ag microflowers decreased by ~60% for E. coli and 63.5% for MRSA compared with that using only Fe₃O₄@SiO₂@Ag microflowers. Thus, we think the effectiveness of vancomycin-modified Fe₃O₄@SiO₂@Ag microflowers against resistant strains such as MRSA provides a very simple but highly efficient strategy to combat drug-resistant bacterial infections. Moreover, the magnetic property renders the Van/Fe₃O₄@SiO₂@Ag microflowers to be facilely controlled by an orientation magnetic field after antibacterial behavior. The recovered products can then be reused many times. We further demonstrated that the antimicrobial effect of the fabricated Van/Fe₃O₄@SiO₂@Ag microflower was maintained at no less than 90% after cycling for five times, indicating the high stability of the product. Hence, the Van/Fe₃O₄@SiO₂@Ag microflowers are potential effective and recyclable antibacterial agent for medical and environmental applications.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (No 81230089), National High Technology Research and Development Program of China (No 2015AA020929), Beijing Municipal Science & Technology Commission (No Z161100000116040), and Beijing Nova Program (No Z141107001814071).

Disclosure

The authors report no conflicts of interest in this work.

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Supplementary materials

Figure S1 Zeta potential of Fe3O4@SiO2 microspheres in ethanol.

Figure S2 Transmission electron microscopy images of Fe3O4@SiO2–Ag seed synthesized with different concentrations of AgNO3: (A) 0.2, (B) 0.4, and (C) 0.8 mM.

Figure S3 Transmission electron microscopy images of Fe3O4@SiO2@Ag microflowers synthesized (A) without polyvinylpyrrolidone (PVP) and (B) with 1 mg/mL PVP.

Figure S4 Absorption spectra of vancomycin solution (a) before and (b) after binding to 11-mercaptoundecanoic acid-modified Fe3O4@SiO2@Ag microflowers.

Figure S5 Bacterial growth curves are employed to evaluate the antibacterial activities of the Fe3O4@SiO2@Ag microflowers against (A) Escherichia coli and (B) methicillin-resistant Staphylococcus aureus (MRSA). Different concentrations of the Fe3O4@SiO2@Ag microflowers were added to the culture of E. coli and MRSA (10–50 and 20–60 μg mL−1, respectively). The control group was tested in the absence of Fe3O4@SiO2@Ag microflowers. The growth of bacteria was assessed by determining optical density (OD) at a wavelength of 600 nm.

Figure S6 Thermogravimetric analysis (blank line) and differential scanning calorimetry (DSC) (blue line) curves of Van/Fe3O4@SiO2@Ag microflowers (A) before and (B) after five antimicrobial cycles.