Antibacterial Effect of Silver-Incorporated Flake-Shell Nanoparticles

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Antibacterial Effect of Silver-Incorporated Flake-Shell Nanoparticles under Dual-Modality Qin Tang, Jia Liu, Lok Kumar Shrestha, Katsuhiko Ariga, and Qingmin Ji ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02507 • Publication Date (Web): 07 Jul 2016 Downloaded from http://pubs.acs.org on July 7, 2016

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ACS Applied Materials & Interfaces

Antibacterial Effect of Silver-Incorporated Flake-Shell Nanoparticles under Dual-Modality Qin Tang, † Jia Liu,§ Lok Kumar Shrestha,§ Katsuhiko Ariga,§ and Qingmin Ji, †* † Herbert Gleiter Institute of Nanoscience, Nanjing University of Science & Technology, 200 Xiaolingwei, Nanjing 210094, China. § Supermolecules Group, WPI Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science, 11 Namiki, Tsukuba, Ibaraki, 305-0044, Japan. ABSTRACT: Silver has been recognized as a broad-spectrum antimicrobial agent and extensively used in biomedical applications. Through a sequential one-pot synthesis strategy, we have successfully incorporated silver into flake-shell nanoparticles. Due to the simultaneous growth of networked nanostructures of silica and in-situ reduction of silver ions, homogeneously distributed silver into the shell of the nanocapsule was formed. The antibacterial test indicated that the silver-incorporated silica nanocapsule exhibits effective antibacterial activity, inhibiting the bacterial growth by 75%. In addition, with the encapsulation of other antibiotic agent into the structure, an enhanced antibacterial effect under dual-modality could also be achieved. Keywords. nanocapsule, silver, antibacterial, silica, drug release

1. INTRODUCTION The antibacterial property of silver (Ag) has been known for over centuries.1-3 With the development of the strategic design of novel nanomaterials or nanostructures,4-8 Ag-based nanomaterials have been recognized as broad-spectrum antimicrobial agent,9-11 which encourage their potential applications in various fields including biomedicine, water purification, food preservation, cosmetics, and clothing manufacturing etc.12-16 The bactericidal action of Ag-based nanomaterials mainly relies on the release of silver ions.17-19 In order to maintain the antibacterial efficacy, Ag needs supporting matrices to keep their morphological features.20-22 Therefore, the design of novel Ag-incorporated nanostructures is essential to open newer possibilities of this antibacterial agent. Until now, various synthetic strategies have been reported for the production of Ag-incorporated nanomaterials. Ag salts or Ag nanoparticles can be integrated or incorporated into various inert matrices such as polymers,23-25 supramolecular structures,26-28 ceramics,29,30 and other inorganic porous structures.31-34 It should be noted that the stabilization of Ag Scheme 1. (a) Sequential one-pot fabrication of silica nanocapsule with nanostructures in the support matrix is extremely important Ag embedded inside the shells. (b) Enhanced antibacterial effect by Agfrom the view point of safety and performance. Unexpected incorporated nanocapsule with or without drug loading (dual-modality). exposure to the external media or leakage of silver from the matrix surface, not only affect the antibacterial efficacy, but antibacterial drugs from natural and friendly materials have also lead to the environmental and health safety risks.35,36 attracted much attention for acting as promising alternatives in Therefore, development of novel synthetic methods for the recent years.42,43 Catechin, a natural polyphenol, can be fabrication of stable Ag-based nanocomposite structure, which extracted from green tea or cocoa. It is a non-toxic, cheaper can prevent uncontrollable growth, aggregation, and escape of drugs that may be effective for a number of health issues, such Ag nanostructures, is still a key challenge in this field. as reduction of cholesterol, protection against cancer, On the other hand, antibacterial drugs including antibiotics, antioxidation and antimicrobial activity.44,45 Its antibacterial quaternary ammonium or phosphonium compounds etc. are effect has also been reported to protect against oral, intestinal, also being widely used to effectively protect the public food-borne bacteria and anti-toxicity activity for various health.37-40 However, these drugs may associate with the bacterial haemolysins.46 As this phenolic compound can be concerns of antibiotic resistance, complex synthesis susceptible by oxidation and condensation between procedures, environmental pollution and high cost.41 Therefore, catechins,47,48 it is essential to protect them into solid supports ACS Paragon Plus Environment


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especially during the long-term and repeated usage. The mesoporous structures and solid nanoparticles have been successfully used as host materials for the adsorption or immobilization of the phenolic compounds.49-51 Herein, we present a sequential one-pot synthesis process for the construction of novel silica nanocapsule with Ag embedded inside the shells (Scheme 1a). In our previous work, we have succeeded to design silica nanocapsules with various complex shell morphologies under a spontaneous regrowth process, which showed excellent proteins and nanoparticles immobilization capability.52-55 Since the reaction occurs in aqueous solution, we anticipate that Ag ions can be easily introduced during the process and form Ag nanocrystals with regrown silica nanostructures, which may lead to a novel nanocomposite with superior morphology. As Ag is in-situ formed during the regrowth of the network of silica flakes, the uniform incorporation and fixation of Ag in silica can be easily achieved compared to other cases, which use the postformation of silica layers on the surface of Ag nanostructures to avoid the leakage.56,57 The post-formation of silica layers on Ag nanostructures always turn out to be relatively thick (thickness >50 nm), which may defer the immigration of Ag ions to exert the antibacterial effect.58,59 Very thin (around 10 nm) silica flakes with the open network structure in our case should be beneficial to transfer Ag ions to external. The antibacterial test on this nanocomposite structure showed the inhibition efficacy of bacterial growth by 75%，according to the comparison on the colony numbers in the medium with and without the Ag-incorporated nanocapsules. Additionally, the hollow interior of the nanocapsule allows further loading of antibacterial agents. With the post-encapsulation of catechin in the nanocapsule, an enhanced antibacterial effect was achieved and can be prolonged for at least 5 days. Direct incorporation of Ag in the capsule structure not only effectively protects Ag from aggregation, also provides a more easily accessible porous network for loading and releasing drugs to bacteria, demonstrating that Ag embedded flake-shell nanoparticle would be novel Ag-based nanocomposite structure for antibacterial under dual-modality system (Scheme 1b).

2. EXPERIMENTAL SECTION 2.1 Materials. Silica colloidal solution with an average particle size of 500 nm was provided by Nissan Kagaku Company (Japan). Silica particles were collected from the solution by centrifugation and then heated at 500 oC for 5 hrs. Silver nitrate (AgNO3) and (+)catechin were purchased from Wako Pure Chemical Industries, Tokyo Japan. Sodium borohydride (NaBH4) was purchased from Sigma-Aldrich. All reagents were used as received. Water used in the experiments was purified through a Purelab Prima system to a resistivity of 18.2 MΩ cm. 2.2 Synthesis of silver-incorporated silica nanocapsules. Silica particles (200 mg) were added into a 50 mL capacity glass bottle containing 10 mL water. After sonication for 30 min, 1 mL AgNO3 aqueous solution (1 mM) was added and shaken using a mechanical shaker for 24 hrs at 25 oC. The mixture were collected by centrifugation and washed 3 times with water.

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The Ag ions pre-coated silica particles were dispersed in 5 mL of NaBH4 aqueous solution (0.1 g/L) and then heated at 75 oC in a 20 mL teflon-lined autoclave for 20 hrs. The sample was collected by centrifugation and washed several times with pure water until the washings achieve a neutral pH. The resulted nanocapsules were freeze-dried. 2.3 Encapsulation of drug molecule in the nanocapsules. Dry nanocapsules (5 mg) were added in catechin solution (1 mg/mL) for drug loading. After shaking for 2 hrs at room temperature, the dispersion was left undisturbed for overnight. The samples were collected by centrifugation and dried under vacuum. Thermogravimetric analysis was used for the determination of the quantity of catechin loaded in the particles. For the releasing of catechin from nanocapsules, dry samples were dispersed into 3 mL water. Periodically, a 1 mL aliquot was drawn from the medium to follow catechin release then placed back in the same vessel in order to maintain the volume constant. The released amounts of catechin were determined by optical absorbance at 280 nm.60 2.4 Antibacterial test for the nanocapsules. The antibacterial capability of the samples was measured by using standard count plates. The testing water (I) was taken from watercourse and placed in glass bottle for 3 days. The samples (1 mg) were mixed into the testing water (1 mL). The dispersions were kept for incubation at room temperature for 15 days. For the detection procedures, commercially available checker contact plates (total count plate, DENKA SEIKEN Co Ltd, Japan) were activated at 37 oC for 24 hrs. The sample solutions (10 µL) were dropped into the plates and cultivated at 37 oC for 24 hrs to grow the bacteria (general bacteria). To visualize the bacteria coverage on surface, 45 drops of (red indicator dye) color developer solution was added onto the surface of agar surface, results development of red color for the colonies. The test was repeated three times to ensure the repetitiveness. The antibacterial effect was compared based on the ratio of the covered and uncovered areas of bacteria on agar surface, using Image analysis software, ImageJ. 2.5 Estimation of antibacterial efficacy. The inhibition efficacy to bacteria growth was estimated by counting the colonies on the surface of the culture medium, using bacteria colony detecting plate (Dayuan Oasis Food Safety Technology co. Ltd.).60 The colony number of the (bare) testing water is treated as a reference value for antibacterial effect, that is the efficacy of the testing water is 0%. The decreasing percent of the colony number in the sample solution in contrast to the bare solution at 24hrs cultivation is calculated as the antibacterial efficacy.60 The standard deviation (SD) is based on five independent experiments. 2.6 Characterizations. Scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM) images were obtained using a Hitachi S-4800 field-emission scanning electron microscope operating at 10 kV and 30 kV, respectively. High resolution transmission electron microscopy (HR-TEM) was performed by using a JEOL-JEM 2100F operating at 200 kV. Zeta potentials were measured on a Zarasizer Nano ZS90 particle analyzer (Malvern Instruments, UK). XRD spectra of the powder samples were measured at 25 °C on a Rigaku diffractometer (RINT 2200) using graded

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d-space elliptical side-by-side multilayer optics and monochromated CuKradiation (40kV, 40 mA). Nitrogen adsorption-desorption measurements were conducted on powdered samples at 77 K on an Autosorb1 surface area and pore size analyzer (Quantachrome Instruments, USA). The specific surface area was calculated from the adsorption branch using the Brunauer-Emmett-Teller model (BET) method. Thermogravimetric analysis (TGA) measurements were performed on a SII TG/DTA 6200 at a heating rate of 5 oC/min. Absorption spectra were recorded using a Shimadzu U-3600 at room temperature. FTIR spectra were measured on a Nicolet Nexus 670 FTIR instrument.

3. RESULTS AND DISCUSSION Fabrication of silver-incorporated silica nanocapsule. The Ag-incorporated silica nanocapsule (Ag-SiNCap) is prepared following a sequential one-pot synthesis strategy. Ag ions were pre-coated on surface of silica particles by mixing AgNO3 solution into the dispersion of silica particles. The negatively charged silica can direct the adsorption of the positively Ag ions on its surface. The zeta-potential measurement indicated that the surface charge of the silica particles is reduced from -29 to -24 mV after adsorption of Ag ions. The particles were then reacted in NaBH4 solution at 75 oC for 20 hrs. Strongly basic NaBH first dissolves silica as 4 monosilicate, then supersaturation of silicate in the vicinity of the particles induces regrowth with the aid of BO2 generated from NaBH4.52,53 Through this hydrothermal dissolutionregrowth process, solid silica particles dissolve slowly with concomitant growth of the silicate structures on the surface, forming networked silica flakes as shell. In the meantime, the Ag ions on the surface were also reduced into Ag during this process.

Figure 1. SEM/TEM images of (a) and (b) silica nanocapsules without Ag; (c) and (d) Ag-incorporated silica nanocapsules.

The Ag-SiNCaps display light brown color due to the existence of Ag nanoparticle (Inset Figure 1c). While the sample prepared without Ag ions appears (SiNCaps) as white powder (Inset Figure 1a). Figure 1c (SEM) and Figure 1d (TEM) show the morphology of the Ag-SiNCaps. The obtained nanocapsules have uniform diameter ( ～ 600 nm)

Figure 2. (a) and (b) TEM images of Ag-incorporated silica nanocapsules at high magnification. (c) EDX spectrum of Ag-incorporated silica nanocapsules.

with the flake shell of ～80 nm, and the flake thickness of ～ 10 nm. The nanosheet morphology of Ag-SiNCaps seems to be more robust than SiNCaps, which is formed into relatively “soft” network. Compared to the Ag-SiNCaps, the flakes of SiNCaps are thinner, ca. 5 nm (Figure 1a and 1b). HR-TEM images reveal the distribution of Ag nanoparticles and its crystalline lattice in the amorphous silica structure (Figure 2a, 2b and S1). The energy-dispersive X-ray (EDX) analysis of the Ag-SiNCaps further confirmed the presence of Ag in the structure (Figure 2c). Based on the element analysis result, the weight ratio of Ag to Si is estimated to be 0.06, approximately 1.3wt% of Ag is present in the nanocapsules. The structure of Ag was also further confirmed by powder Xray diffraction (XRD) (Figure S2). XRD reflections at diffraction angles of 38o, 45o degrees correspond to the (111) and (200) planes of the face-centered cubic (fcc) phase of Ag. UV-Vis absorption spectrum also showed a broad plasmon resonance band of Ag centered around 400 nm (Figure S3). We have tested the fixation of Ag nanoparticles on the silica flakes using UV-Vis spectrometry. The Ag-SiNCaps was dispersed in water for a month and subsequently UV-Vis spectrum of the supernatant phase was recorded. We did not see any signature of Ag nanoparticles in the supernatant solution from the UV-Vis spectrum (Figure S4), which means that no leakage of Ag nanoparticles from Ag-SiNCaps occurs upon long-term storage. Furthermore, we also did not observe any aggregates of Ag nanoparticles in TEM (Figure S5). From these observations, it can be concluded that this synthesis method facilitate the fixation of Ag inside the silica network. Drug encapsulation in silver-incorporated silica nanocapsule. The nanocapsules with the flake shell morphology provided more easy accessible surface and hollow interior space for binding and encapsulation of the adsorbent



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molecules. Nitrogen adsorption analysis indicated that the AgSiNCaps possess surface area of 509 m2/g and pore volume of 0.915 cm3/g. The values are a little lower than SiNCaps, which are 695 m2/g and 1.27 cm3/g. We suppose that the decrease in surface area and pore volume is due to a result of the embedding of Ag nanoparticles inside the mesopores of silica framework.

Figure 3. (a) Thermogravimetric analysis (TGA) of catechin-loaded nanocapsules. (b) Cumulative catechin release (25 oC) from the nanocapsules in water. (i) silica nanocapsules without Ag; (ii) Agincorporated silica nanocapsules.

The encapsulation of guest molecules in the Ag-SiNCaps was investigated by loading with catechin. The dispersion of Ag-SiNCaps (1 mg/mL) was mixed with the catechin solution (1 mg/mL) and kept undisturbed for overnight at room temperature. TGA analysis showed that around 14 wt% of catechin was encapsulated inside the Ag-SiNCaps (Figure 3a), which is estimated from the weight loss above 120 oC. This value is a little higher than SiNCaps (without Ag), which is about 10 wt% (Figure 3a). It should be noted that the formation process of Ag-SiNCaps does not involve any organic reagents. Thus, the weight loss above 120 oC should mainly originate from the encapsulated molecules in the nanocapsules. The encapsulation of catechin in the nanocapsules can also further be confirmed by FTIR spectra (Figure S6). The bands due to Si-OH bending (1650 cm-1) and stretching (3500 cm-1) vibrations showed a red shift and strengthened after loading with catechins. The appearance of new bands from 1100-1700 cm-1 and 3000 cm-1 can be assignable to CO, C=C, C=O, CH vibrations from the aromatic rings of catechins, which indicates the presence of catechins in the nanocapsules. The presence of Ag in the silica nanocapsule structure may facilitate the adsorption of catechin also based on -electron

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interaction, instead of only hydrogen bonding between silica and catechin. The significant shift at the peak around 1650cm-1 for Ag-SiNCaps can be due to the presence of -electron interaction in the structure. Thus, a sustained release behavior is observed for the Ag-SiNCaps compared with SiNCaps. As it can be seen in Figure 3b, SiNCaps reach the equilibrium state of releasing relatively faster compared to Ag-SiNCaps. During the release process of catechin from Ag-SiNCaps, no obvious releasing of Ag was observed. We can not find adsorption around 400 nm for Ag nanoparticles in the UV-Vis spectra from the supernatant solutions of the catechin-loaded AgSiNCaps at different releasing time (Figure S7). It is also worth to mention that the nanocapsules have shown well capability to prohibit catechin from oxidation. The fresh catechin solution is in light yellow color. It will become dark color due to the oxidation, which limits its antibacterial applications.47 As shown in Figure S8, the color of catechin solution changed into dark orange when kept at room temperature for 3 days, while the colors of the dispersions of catechin-loaded nanocapsules remain as light yellow. Especially for Ag-SiNCaps, the dispersion almost kept same color as the fresh catechin solution. This result implies that a prolonged antibacterial effect from catechin may be achieved by its encapsulation in the nanocapsules. Antibacterial effect of silver-incorporated silica nanocapsule. The antibacterial effect of Ag-SiNCaps was studied by checker contact plates for all colonies growth. We have prepared the testing water (I) from watercourse, which is considered to have bacteria inside. We have used Coli-form detecting plate and Streptococcus detecting plate to confirm the types of bacteria in the testing water (I) (10 days). It indicated that there are Coli bacteria in the testing water (colony number is around 3×103 CFU/mL). While the Streptococcus detection showed negative result. We may estimate that the bacteria in the testing water (I) mainly are the gram-negative bacteria. After mixing the testing water (I) with nanocapsules for 1 day, we spread the mixture on the agar surface of the checker plate and incubated at 37 oC for 24 hrs. Bacteria grown on the

Figure 4. Checker plates (with agar) supplemented with (a) the testing water (I) (bare), and the testing water (I) containing (b) silica nanocapsules and (c) Ag incorporated silica nanocapsules after incubation 24 hrs. (d) the calculated average coverage ratios of bacteria on the agar surface of the checker plates (a), (b) and (c).

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Table 1. Antibacterial activity of catechin, silica nanoparticles and nanocapsules with or without catachin. Incubation time a

No. of colony (CFU/mL) b

(hrs)

Bare (the testing water)

Catechin

Si-NP (500nm)

SiNCap

AgSiNCap

Catechin-loaded SiNCap

Catechin-loaded AgSiNCap

24

1.5×104

9×103

3.5×104

9.1×103

3.8×103

8.3×103

1.4×103

72

8.2×10

3.4×10

6.7×10

3.7×10

2.1×10

1.6×10

< 30

120

>9×105

5.5×105

8.4×105

5.7×105

< 30

5.5×105

300

Antibacterial efficacy (%) c

-

40

19d

39

75

45

91

4

4

4

4

3

4

a

The incubation time means the mixing time of the testing water (I) with sample solutions at room temperature. The values were counted by the bacteria spot larger than 0.1mm on the checker plate after 24hrs cultivation at 37 oC. c The standard deviation (SD) is less than5. d The efficacy was calculated based on the decreasing percent of the colony numbers in sample solution in contrast to testing water (I) after 72hrs incubation. b

agar surface was visualized through the red indicator color. The antibacterial effect of Ag-SiNCaps was estimated by counting the colonies grown on the surface of detecting plates after cultivation. As shown in Figure 4 and Table 1, the testing solution (bare) with bacteria displayed more than 104 CFU/mL average density of bacteria. We directly compared the antibacterial effect from the images of checker plates with bare and nanocapsules with or without Ag incorporated. For bare solution, the agar surface of checker plate was almost fully covered with bacteria (about 81%). The growth of bacteria was much restrained in cases of mixing with nanocapsules. The surface coverage of bacteria is 52% for SiNCaps and 21% for Ag-SiNCaps, respectively. Note that even without Ag, the nanocapsule itself showed antibacterial effect. We anticipate that trace of salt ions in the nanocapsule and also the flake morphology of the nanocapsule may hinder or delay the bacteria growth. This anticipation was further confirmed by checking the antibacterial effect of silica nanoparticles (SiNPs) with 500 nm in diameter (Figure S9), which has similar size to the flake-shell nanoparticles, but with smooth surface. The result indicated that the SiNPs have very weak antibacterial effect (Table 1). With the incorporation of Ag inside the structure, stronger inhibition effect on bacterial growth is observed. The number of colonies from the medium with Ag-SiNCaps is significantly reduced in comparison to the cases of the bare solution, SiNP, and SiNCaps (Table 1). The antibacterial efficacy of AgSiNCaps can also be enhanced with increasing the amount of the nanocapsules in the testing dispersion (Table S1). Besides relying on Ag in the nanocapsule to enhance the antibacterial activity, the inhibition efficacy of nanocapsules to bacterial growth could further be improved by loading catechin inside the nanocapsule. The inhibition of the bacterial growth was nearly completed for catechin-loaded Ag-SiNCaps (Figure 5). This significantly superior antibacterial effect can also be maintained for several days due to the combined effect from catechin and Ag (Figure 5 and Table 1). Although catechin is antibacterial effective in compared with the bare solution and silica particles, it not showed high antibacterial activity as the cases when loaded in the Ag-SiNCaps. Its activity also seems to be declining after 3 days. We suggest that this might be due to the oxidation of catechin. It has been reported that hydroxyl groups in the phenolic compounds are the key factors to the antibacterial activity.61 The antibacterial activity may become weak with reducing the number of hydroxyl groups. Phenols in catechin can be ionized into

Figure 5. Checker plates (with agar) supplemented with the testing water (I) containing catechin encapsulated Ag-incorporated silica nanocapsules after incubation (a) 12 hrs and (b) 72 hrs.

phenoxide ion in aqueous solution and oxidized to be aromatic ketone (Scheme S1).62,63 The color change of catechin solution from light yellow to dark orange during the incubation with the testing water indicates the occurrence of the oxidation. The UV-Vis spectrometry can further confirm the oxidation of catechin. The UV-Vis spectrum of fresh catechin solution shows a strong adsorption peak at 280 nm, which is completely disappeared in the UV-Vis spectrum of the catechin solution after mixing with the testing water for days. And instead a new broad peak centered at 380 nm appeared, which can be ascribed to the formation of quinones from phenols of catechin (Figure S10). As Ag-SiNCaps still exhibited relatively high antibacterial effect even under lower concentrations (Table S1), we suggest that the incorporation of Ag plays a major role on bacterial inhibition. The releasing of catechin from the nanocapsules will mainly enhance the short-term antibacterial effect. Upon increasing the amount of catechin in the testing water or from the comparison of SiNCap and catechin-loaded SiNCaps at different addition amounts, we can find that the antibacterial effect might be strengthened to some extent by catechin (Table S1). However, besides oxidation, the condensation between catechins might also lead to a decline of the antibacterial activity with time. Thus, larger addition amount of catechin may not result into a better performance of antibacteria. It should be noted that when catechin is dissolved in pure water and stored in refrigerator, the solution is stable for at least 1 month (Figure S10). It implies that catechin may keep its activity under certain conditions, for example the maintenance on the clean states.



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In addition, we found that the bacteria in the testing water might promote the oxidation of catechin, as the color change became faster in the testing water than that of in pure water at the same condition. This phenomenon of catechin might be used for acting as an indicator to detect the presence of bacteria in the solution. As the testing water (I) only have gram-negative bacteria, to further prove the inhibitory activity of the nanocapsules for broad range of bacteria, we also checked another testing water. The testing water (II) is collected from fishpond, which contains both Coli and Streptococcus bacteria. The detection results indicated that the colony number of Coli and Streptococcus bacteria in the testing water (II) is around 7.8×104 CFU/mL and 3.1×104 CFU/mL, respectively. After mixing with nanocapsules, we find that similar effective inhibition of bacteria growth can also be achieved (Table S2). In compared with the efficacy to the testing water (I), catechin or the catechin-loaded nanocapsules showed an enhanced antibacterial effect on the testing water (II). It might imply that catechin is more effective on the gram-positive bacteria. From the above results, we confirm that the Agincorporated flake-shell nanoparticles can successfully bring antibacterial activity under dual-modality, that is generation Ag ion and releasing antibacterial reagent. The incorporation of Ag ensures a long-term bacterial inhibition effect. The encapsulation of antibacterial reagent plays a more active role on the short-term effect. Since the nanocapsules can be loaded with various molecules, other antibiotics or specific reagents may also be used according to the requirements of applications.

4. CONCLUSIONS In this paper, we have described the construction of Agincorporated silica nanocapsules with flake-shell morphology, and demonstrated their antibacterial effect under dualmodality. Through the sequential one-pot synthesis strategy, Ag is facilely embedded inside the silica network and formed a nanostructured hollow morphology. The composite nanocapsules exhibited an enhanced antibacterial activity due to the incorporation of Ag. In addition, the hollow morphology and large surface area enabled the loading and protection of natural antibiotic reagent in the composite nanocapsules, and substantially increased the inhibition efficacy of bacterial growth. This Ag-incorporated nanocapsule may act as a promising antibacterial structure or additive in various applications, for example filters, coatings or sensors. Besides incorporation of Ag into the nanocapsule structure, the formation process may also possibly be applied for other metal-incorporated composite structures. For example, we have succeeded to fabricate Au incorporated nanocapsules by similar procedures (Figure S11). Therefore, the synthesis strategy may also be a versatile synthesis method to obtain novel nanocomposite materials.

AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ASSOCIATED CONTENT Supporting Information. Further experimental information including experimental details for drug release and detection of bacterial growth; additional data for XRD result, UV-Vis spectrum, TEM/SEM images, FT-IR spectra, antibacterial results, etc. “This material is available free of charge via the Internet at http://pubs.acs.org.”

ACKNOWLEDGMENT This work was partly supported by World Premier International Research Center Initiative (WPI Initiative) of Japan Science and Technology Agency (JST). Q.J thanks financial support from Herbert Gleiter Institute of Nanoscience (HGI); from “the Fundamental Research Funds for the Central Universities”, No. 30916015108; Science and Technology Program of Jiangsu Province: BK20151484 and AD41572.

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Antibacterial Effect of Silver-Incorporated Flake-Shell Nanoparticles

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