Antimicrobial Silver Nanoclusters Bearing Biocompatible


Sep 30, 2016 - To improve the biocompatibility of silver antimicrobial agents, we have synthesized thiol-terminated phosphorylcholine (PC-SH)-protecte...
0 downloads 4 Views 1MB Size


Subscriber access provided by HKU Libraries

Article

Antimicrobial silver nanoclusters bearing biocompatible phosphorylcholine based zwitterionic protects Arunee Sangsuwan, Hideya Kawasaki, Yoshinobu Matsumura, and Yasuhiko Iwasaki Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00455 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on October 2, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Antimicrobial silver nanoclusters bearing biocompatible phosphorylcholine based zwitterionic protects

Arunee Sangsuwan1, Hideya Kawasaki2,4 Yoshinobu Matsumura3,4 and Yasuhiko Iwasaki*2,4

1

Graduate School of Science and Engineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka, 564-8680, Japan

2

Department of Chemistry and Materials, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka, 564-8680, Japan, Tel: +81-6-63680090, Fax: +81-6-6368-0090, E-mail: [email protected]

3

Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka, 564-8680, Japan

4

ORDIST, Kansai University 3-3-35 Yamate-cho, Suita-shi, Osaka, 564-0836, Japan

*Corresponding author

ACS Paragon Plus Environment

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Infection is one of the most serious issues in the medical treatments, leading to the development of several antimicrobial agents. In particular, silver ions released from silver substrates is well known as a reliable antimicrobial agent that either kills the microorganisms or inhibits their growth. Unfortunately, many reports have shown that silver-based antimicrobial agents are toxic for human cells as well. To improve biocompatibility of silver antimicrobial agents, we have synthesized thiol-terminated phosphorylcholine (PC-SH) protected silver nanoclusters (PC-AgNCs) via strong thiol–metal coordination with controlled ultrasmall size of the clusters. A change in plasmon-like optical absorption was studied to affirm the successful synthesis of small thiolated AgNCs through the absorption spectra that become molecular-like for the AgNCs. We observed that PC-AgNCs were spherical with an average diameter of <2 nm. The ultrasmall size clusters were exceedingly immobilized by the PC-SH on the surface, resulting in excellent biocompatibility and antibacterial activity simultaneously. The biocompatible/antimicrobial PC-AgNCs exhibit interesting advantages compared with other silver antimicrobial agents for medical application.

Keywords: silver nanocluster, antibacterial activity, biocompatibiity, 2-methacryloyloxyethyl phosphorylcholine, thiol-ene reaction

INTRODUCTION Metallic silver (Ag) has the potential to deliver a reliable inorganic nanomaterial with a high antimicrobial activity.1,

2

It was reported that the antibacterial activity is influenced by

various factors such as the nanoparticle size,3 types of the ligand, and different types of bacteria.4

ACS Paragon Plus Environment

Page 2 of 22

Page 3 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

The antimicrobial mechanism of the silver metal has been proposed to proceed via the direct damage of the bacterial cell membrane,1 where the silver metal can react with the sulfurcontaining proteins on the cell membrane leading to the disruption and death of the bacteria.5 In addition, reactive oxygen species (ROS) and silver ions arising from the silver metal can interrupt important DNA-related processes such as DNA replication, contributing to cell toxicity.5 Owing to the affinity of silver for the bacterial cell membrane, silver metal shows inhibitory activity in both Gram-negative and Gram-positive bacteria.6 For positively charged Ag nanoparticles (AgNPs), inhibitory activity is provided by the electrostatic attraction between the negatively charged bacterial cells and the positively charged nanoparticles as well as the reactions of the AgNPs with sulfur-containing amino acids in the bacterial cell wall.

4, 7

In

contrast, negatively charged AgNPs exhibited relatively low toxicity, depending on the zeta potential (ζ/mV) of the nanoparticles. Citrate-AgNPs (ζ = −38 mV) exhibited lower toxicity than H2-AgNPs (ζ = −22 mV) and poly(vinyl pyroridone) (PVP)-AgNPs (ζ = −10 mV) because the increased electrostatic barrier resulted in the reduction of the likelihood of interactions between the cell and the particle, leading to lower toxicity.7 In addition, the cell wall thickness of Gram stain bacteria is a determining factor of antimicrobial activity. The thick multilayer of Grampositive bacteria (15–80 nm thick) such as Staphylococcus aureus can prevent toxicity caused by the particles better than Gram-negative bacteria (10–15 nm) such as Escherichia coli that have only a thin-layer of peptidoglycan, giving rise to a higher toxic effect.8 Particle size is another factor controlling the silver metal antimicrobial activity. It has been reported that AgNPs displayed an obvious size-dependent antibacterial activity.9 Smaller particles easily penetrate into the cell and often show higher toxicity.3 The recently investigated ultrasmall silver nanoclusters (AgNCs) with an average diameter of <2 nm are fascinating owing

ACS Paragon Plus Environment

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 22

to their unique properties such as strong luminescence and unique optical and antimicrobial properties that are unlike those of AgNPs with a larger size (>2 nm) .9,

10

These unique

properties are important not only for antimicrobial activity but also for the use of AgNCs in optical probes in biosensing11 and bioimaging12 in medical diagnosis and treatment. The capping ligands of AgNCs play an important role in their use for biomedical applications; therefore, various capping agents such as polymers1, polyelectrolytes,13 DNA,14, 15 and proteins16 have been employed as the AgNC capping agents. Small AgNCs are more efficient antimicrobial agents than the larger AgNPs owing to their large surface area and small size. Setyawati et al17 reported that the antimicrobial efficacy of glutathione (GSH)-AgNCs depended on the core surface speciation (Ag+ and Ag0 core states) with GSH-Ag0-NCs exhibiting higher cellular toxicity than GSH-Ag+-NCs as a result of the more rapid release of ROS from GSH-Ag0-NCs.17 To enhance the base silver capacity for antimicrobial treatment, the silver should exhibit toxicity specifically for the target microbial cells without damaging the healthy cells. This requirement is referred to as antimicrobial safety. From the standpoint of antimicrobial safety, the surrounding tissue and other organs provide new inspiration for the design of silver nanomaterials with excellent antimicrobial properties. 2-Methacryloyloxyetyl phosphorylcholine (MPC) is a hygroscopic monomer18 that has been used to modify silver nanoparticles (AgNPs) with a size of 13 nm in our previous research.19 The phosphorylcholine (PC)-modified AgNPs (PC-AgNPs) exhibited not only a high stability of PC-AgNPs in the cell culture medium but also a good dispersity and excellent biocompatibility relative to the results obtained in other studies.20, 21

The previous results for PC-AgNPs strongly motivated us to develop more effective

biocompatible silver metal by decreasing the size of the nanoparticles in order to provide an increased surface area to volume ratio for PC immobilization. In this research, we have prepared

ACS Paragon Plus Environment

Page 5 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

AgNCs protected with the biocompatible thiol-terminated phosphorylcholine (PC-AgNCs) in order to obtain reduced toxicity of the particles and less healthy tissue damage, enabling the use of these AgNCs as antibacterial agents. We studied the size control of PC-AgNCs by changing the Ag/MPC molar ratio. Our findings indicate that PC protection of AgNCs improves dispersity, stability as well as biocompatibility of the nanoclusters. Consequently, at a given concentration range, PC-AgNCs exhibited toxicity to the target microbial cells without damaging the healthy cells.

Figure 1. Chemical structure of PC-SH.

RESULTS AND DISCUSSION Characterization of PC-AgNCs. UV–Vis absorption spectroscopy is often used as the first measurement to differentiate AgNCs from their larger counterparts by different peak locations and origins of their optical absorptions.22 While the localized surface plasmon resonance peak of AgNPs is generally located at 400 nm, this peak is not observed in the absorption spectra of ultrasmall AgNCs (<2 nm). The continuous density of states breaks up into discrete energy levels, leading to the presence of several distinct absorption peaks in the UV–Vis region, as shown in previous studies.22,23 Chakraborty et al. confirmed the transformation from molecularlike absorptions in the UV–Vis absorption spectra to plasmonic-like signals around 460 nm between the small thiolated AgNCs with <114 Ag atoms and large AgNPs with >150 Ag atoms.21 This transformation was also observed in the present study. As the molar ratio of Ag

ACS Paragon Plus Environment

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ions to PC-SH (Figure 1) at the synthesis of PC-AgNCs increased, the absorption spectra changed from plasmonic-like absorptions (1:0.5 and 1:1) around 460 nm to molecular-like absorption with multiple peaks (400, 480, and 650 nm) (1:2.5 and 1:5), as shown in Figure 2. This suggests the transformation of large PC-AgNPs into small PC-AgNCs with the molar ratio of PC-ligand. Dynamic light scattering (DLS) supported the formation of small PC-AgNCs, and the number averaged hydrodynamic diameters of PC-AgNCs with the ratio 1:2.5 was 2.5 ± 0.7 nm (Supporting Figure S1). The TEM images also showed reduction in size as the molar ratio of PC-SH at the synthesis of AgNCs increases (Figure 3): 4.9 ± 2.9 nm for 1:0.5 and 3.3 ± 0.9 nm for 1:2.5). The larger size estimation for AgNCs by TEM may be attributed to the fusion of the AgNCs via the high-energy electron beam used in TEM observation. To determine the formula of PC-AgNCs, we conducted ESI-MS of the NCs, as shown in Figure 4. The ESI mass spectrum showed the shape peak at m/z =1213.05), consistent with the theoretical m/z value of 1213.04 for Ag3(MPC-S)2+. However, we were unable to definitively identify these clusters as Ag3(MPC)2 owing to the susceptibility of these species to fragmentation during mass analysis. According to the TGA analysis, the molar ratio of Ag:PC-ligand in PC-AgNCs which were synthesized with the feed ratio of Ag:PC-SH = 1:2.5, was estimated 1:0.5 (Supporting Figure S2). FTIR spectroscopy provides information about the functional groups on the metal NC surface. In particular, the binding of thiolate ligands to the Ag core in thiolated AgNCs is evident from the disappearance of the characteristic S–H peaks.19 Figure 5 shows the FT-IR spectra of PC-SH and PC-AgNCs, demonstrating the disappearance of the characteristic S–H peaks at 2570 cm−1 for PC-AgNCs and supporting the formation of thiolated PC-AgNCs. The stability of nanoclusters in water was investigated by using UV–Vis absorption spectroscopy. Although the spectra were slightly changed with soaking periods, the distinct multiple peaks of AgNCs were

ACS Paragon Plus Environment

Page 6 of 22

Page 7 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

preserved even after 3 days of incubation (Supporting Figure S3).

Figure 2. UV–Vis spectra of PC-AgNCs. Molar ratios of Ag ions to PC-SH at the synthesis of PC-AgNCs are shown in the figure.

Figure 3. TEM images and size distribution of PC-AgNCs for Ag:PC-SH ratio of (a),(b) 1:0.5 and (c),(d) 1:2.5.

ACS Paragon Plus Environment

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. ESI-mass spectrum of PC-AgNCs (Ag:PC-SH = 1:2.5).

Figure 5. FT-IR spectra of PC-SH and PC-AgNCs (Ag:PC-SH = 1:2.5).

ACS Paragon Plus Environment

Page 8 of 22

Page 9 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Cell viability test. Cell viability of PC-AgNCs was determined by cell counting kit−8 (CCK-8) after the treatment for 24 h. PC-AgNCs exhibited excellent biocompatibility up to the high concentration of 100 µg/mL AgNCs. Even at 200 µg/mL PC-AgNCs, the cell viability was still higher than 60%, as shown in Figure 6. This is consistent with the observation of the cell morphology using a microscope. At 100 µg/mL AgNCs, all cell morphology was fine with no significant toxicity (Figure 6b); the cell morphology was somewhat altered at 200 µg/mL AgNCs (Figure 6c). These cell viability tests demonstrates the low cytotoxicity of PC-AgNCs, compared with other silver nanometals such as bare-AgNPs (3-nm sized particle, 10 µg/mL of particles leading to 30% decrease of cell viability),24 the glutathione-Ag0-NCs (1.5-nm sized particle, 107 µg/mL of particles leading to 53% decrease of cell viability),17 and polyvinylpyrrolidone-AgNPs (3-nm sized particle, IC50 = 0.9–3.2 µg/mL depending on cell type)21. The zwitterionic MPC is inspired by the structure of the cell membrane and has been used in biomedical applications owing to its excellent biocompatibility, protein adsorption, and the ability to resist cell adhesion.18, 19, 25 Owing to the high surface to volume ratio of PC-AgNCs, the excess immobilized MPC on the surface of the NCs displays excellent performance, i.e., low cytotoxicity.

ACS Paragon Plus Environment

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 22

Figure 6. Viability of L929 cells in contact with PC-AgNCs. a) Original-morphology L929 cells; b) Cell morphology after treatment with PC-AgNCs as 100µg/mL; c) 200 µg/mL, respectively; d) Cytotoxicity test of L929 cells treated PC-AgNCs for 24 h at various concentrations. Each value represents the mean of three measurements with standard deviation shown by the bars. Scale bar: 100 µm.

Antimicrobial activity of PC-AgNCs. The antibacterial activity of PC-AgNCs was determined using the growth curve analysis for both Gram positive and negative bacteria, where the dose of PC-AgNCs (0, 20, 50, 100, and 200 µg/mL) was varied with the sequential treatment (Figure 7). The PC-AgNCs can effectively inhibit the growth of both types of bacteria even at a low concentration of 20 µg/mL. Complete inhibition of bacteria growth was achieved at the high PCAgNCs concentration of 100 µg/mL as is evident from the absence of a significant density

ACS Paragon Plus Environment

Page 11 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

increase of E.coli and S.aureus in the culture medium. It is important to emphasize that no cytotoxicity on normal cells was observed for a high concentration of 100 µg/mL. This indicates that PC-AgNCs satisfy antimicrobial safety and only exhibit toxicity for the target microbial cells without damaging the healthy cells in the concentration range of 20 to 100 µg/mL. Moreover, the direct SEM observation of the microorganisms strongly supported the bactericidal effect of PC-AgNCs. Many microorganisms after incubation at 8 h in the absence of the PCAgNCs were observed on the glass surface as shown in Figures 8a and 8c for E.coli and S. aureus, respectively. In contrast, both bacteria types were not observed on the glass surface in the presence of 100 µg/mL PC-AgNCs, as seen in Figures 8b and 8d. Several studies of silver metal in the literature have reported a wide-antimicrobial activity (both Gram stain, fungi, virus, and anti-biofilm)21, 26 with a more toxic response for a broad range of activities that is higher than that of cationic polymers such as chitosan. In the case of cationic polymers, the antibacterial activity was suggested to originate from the electrostatic interaction between the cationic polymer and the negatively charged microbial cell membranes leading to the lesser sensitivity of Gram negative strain to cationic polymers.27 The direct damage of cell membranes resulting in the inhibiting the potent bactericidal activity is known as the bactericidal mechanism.5 Moreover, ROS and Ag+ generated from the silver metal can interrupt the important DNA-based processes such as DNA replication, promoting cell toxicity.5,

28

Such

electrostatic interaction between PC-AgNCs and the negatively charged microbial cell membranes should be weak owing to the zwitterionic nature of PC ligands. To clarify the antimicrobial mechanism for PC-AgNCs, the amount of Ag+ released from PC-AgNCs was determined. The concentrations of Ag+ released from the 100 µg/mL PC-AgNCs suspension after 1 and 3 days of incubation were 0.321 and 0.375 µg/mL, respectively (Supporting Figure S4).

ACS Paragon Plus Environment

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The release of Ag+ would be the dominant factor of the bacterial killing effect of PC-AgNCs because recent studies have indicated that the Ag+ concentration at 0.3 µg/mL showed completely inhibited bacteria

29

and this concentration did not show any adverse effect on the

viability human cells. 30, 31 Considerable differences exist between mammalian cells and the microorganisms that may affect the interaction of the nanoparticles with the cell wall. Mammalian cells have membrane-bound organelles with complex structures with endocytosis allowing the selective passage of certain substances.32 The MPC structure mimics the outer membrane of the mammalian cell and the chemical functionality arising from the phospholipids in the cell membrane structure. It has been reported that MPC homopolymers are inert to nonselective cellular uptake25,33 and nonspecific adhesion.34 This intrinsic nature of MPC endows the PCAgNCs with low cytotoxicity. While the cell wall of the microorganisms consists of a rigid structure,35 a cytoplasmic membrane with a porous channel can take up the nanoparticles with a diameter of up to 5 nm,36 possibly indicating that it is easier for the ultrasmall PC-AgNCs to enter into the membrane of a bacteria than into a mammalian cell. There are reports in the literature that silver metal nanoparticles with a mean size of 5 ± 2 nm were observed in the bacteria membrane and inside cells.5

ACS Paragon Plus Environment

Page 12 of 22

Page 13 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Figure 7. Growth curves of E.coli (left) and S.aureus (right) in the presence of PC-AgNCs effect on bacteria at different concentrations of the clusters.

ACS Paragon Plus Environment

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 22

Figure 8. SEM micrograph of E.coli (a) non-treated and (b) treated with 100 µg/mL of PCAgNCs at 8 h (magnification × 2000). SEM micrograph of S. aureus (c) non-treated and (d) treated with 100 µg/mL of PC-AgNCs at 8 h (magnification × 2000). Scale bar: 10 µm.

CONCLUSION We have synthesized PC-AgNCs via strong thiol–metal coordination, with the control of the size achieved by varying the molar ratio between the PC-SH ligands and Ag+ (AgNO3) at the synthesis. A change from plasmon-like optical absorption of AgNPs to the molecular-like absorption of AgNCs was observed with increasing molar ratio of MPC. PC-AgNCs were characterized by TEM, DLS, FT-IR, and ESI-MS. Owing to the conjugate effect of the membrane-mimicking MPC with ultrasmall AgNCs, we first achieved excellent biocompatibility and antibacterial activity simultaneously for the first time, although generally a trade-off exists

ACS Paragon Plus Environment

Page 15 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

between biocompatibility and antibacterial activity. PC-AgNCs newly developed in this study could be promising high-efficiency antimicrobial agents that are biocompatible with human cells.

MATERIALS AND METHOD Materials. MPC was donated by the NOF Co., Ltd., Tokyo, Japan, and other chemicals of extrapure grades were obtained from Wako Pure Chemical Industries, Ltd., Osaka, Japan; these were employed without further purification. Water was purified using the Millipore Milli-Q system, which involves UV irradiation, ion exchange, and filtration (18.2 MΩ·cm−1).

Synthesis of PC-SH. PC-SH (Figure 1) was synthesized following a previously reported method.37 Briefly, 1,6-hexanedithiol (15.03 g, 100 mmol) and MPC (14.76 g, 50 mmol) were dissolved in a 200-mL round-bottom flask containing 100 mL of degassed chloroform. Then, diisopropylamine (278.8 µL, 2.0 mmol) was added to the mixture and stirred for 22 h at room temperature. The mixture was precipitated into acetone and subsequently dried in a vacuum desiccator for 2 h to eliminate the residual acetone. Finally, the product was dissolved in water and lyophilized. Yied of PC-SH was 88 %.

Synthesis of PC-AgNCs. The clusters were carefully prepared by controlling the Ag:PC-SH (1:0.5, 1:1, 1:2.5, and 1:5) molar ratio. For example, at Ag:PC-SH = 1:2.5, PC-SH (4.6×10−2 mM, 14 mL) and silver nitrate (25 mM, 700 µL) in Milli-Q water were mixed by a magnetic stirrer at room temperature for 1 day under light-shielded conditions; then, sodium borohydride (130 mM, 2 mL) was added dropwise into the stirred solution. The solution was stirred at room temperature for 2 days. The resultant solution mixture was centrifuged at 8,000 rcf for 45 min

ACS Paragon Plus Environment

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 22

using vivaspin-20 tube (membrane; 3,000 MWCO) to eliminate any unreacted PC-SH and AgNO3. Finally, the suspension at the top of the vivaspin-20 tube was lyophilized for 1 day. Clusters using other molar ratios were synthesized following the procedure described above.

Characterization of PC-AgNCs. PC-AgNCs were characterized using a UV–Visible (UV–Vis) spectrophotometer (V-650 spectrophotometer, Jasco, Tokyo, Japan) and electrospray ionization mass spectrometry (ESI-MS, Exactive Plus Orbitrap, Thermo Scientific, West Palm Beach, USA). ESI-MS spectrum of PC-AgNCs was obtained in the positive mode on AgNC solutions (~1 mg/mL) using a mass spectrometer. A dynamic nanospray probe attachment was used, and the spray tip was made from a fused silica capillary. The following settings were used: H2O– MeOH as solvent, 4:1 (v/v); sample flow rate, 3–5 µL/min; capillary temperature, 160 °C; spray voltage, 3.5~4.0 kV. The morphology and size of the particles were then analyzed by transmission electron microscopy (TEM; JEM-1400, JEOL, Tokyo, Japan). TEM samples were prepared by dropping the particles on a TEM grid prior to being dried in a desiccator for one day and observed at 120 kV. Dynamic light scattering (DLS; ZETASIZER NANO-ZS, Malvern Instruments Ltd, Worcestershire, UK) was also used to analyze the average diameter of the particles and the polydispersity index (PDI). The FT-IR spectra of modified particles were recorded in a 500–4000 cm−1 frequency range by an FT-IR spectrometer (FT/IR-6300, Jasco, Tokyo, Japan) in the ATR mode. Thermogravimetric analysis (TGA, Rigaku Corporation, Tokyo, Japan) was performed to determine the ratio of PC-ligand composition on PC-AgNCs.

Cell culture experiment. Mouse L929 cell lines were grown in Dulbecco’s modified Eagle’s Medium (E-MEM; Gibco, life technologies, New York, USA), high glucose medium

ACS Paragon Plus Environment

Page 17 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

supplemented with 10% (v/v), fetal bovine serum (FBS; biowest, Perth, Australia), and 1% antibiotic–antimycotic (Gibco, life technologies, New York, USA). Cells were cultured in a cell culture flask at 37 °C in a humidified atmosphere of air and 5% CO2. During the experiment, a routine subculture was fabricated every 4 days by detaching the cells with a trypsin solution (0.25% trypsin containing 0.01% EDTA) and changing and diluting the medium at 2.5 × 104 cells/mL. To determine the viability of the cells in contact with PC-AgNCs, a Cell Counting Kit8 (CCK-8, Dojindo, Kumamoto, Japan) was used. L929 cells (1.0 × 105 cells/mL, 100 µL) were seeded in a culture medium in 96-well plates and were then incubated for 24 h at 37 °C in a 5% CO2 incubator. Then, the cells were treated with PC-AgNCs at various concentrations (0–200 µg/mL) followed by incubation for 24 h. Meanwhile, Control A (cells and a culture medium), Control B (culture medium only), and Blank (particles in a culture medium) samples were characterized under identical conditions. After incubation, the morphology of the cells was observed by microscopy. CCK-8 reagent (10 µL) was added into each well, and the cells were incubated in the dark for 4 h at 37 °C followed by the measurement of absorbance at 450 nm using a microplate reader. To ensure the reproducibility of the results, the experiment was performed in triplicate. The percentage cell viability was calculated according to (1):

Cell viability (%) = (Sample − Blank)/(Control A − Control B) × 100

···(1)

Antimicrobial test. To examine the antibacterial property of PC-AgNCs, Escherichia coli OW6 (E. coli)38 and Staphylococcus aureus Mu50 (S. aureus)39 were used in this study. The bacteria were grown in 5 ml of Todd Hewitt broth containing 0.1% Bacto yeast extract (THY) medium with a concentation of PC-AgNCs (0, 20, 50, 100 and 200 µg/mL) different from that of the

ACS Paragon Plus Environment

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 22

control without PC-AgNCs. Growth or killing rates and bacterial density were determined by measuring OD at 600 nm using a UV–Vis spectrometer. The OD values are related to the bacteria density, which for the bacteria in contact with PC-AgNCs can be graphically described as growth curves.40 The bacteria suspensions, no-treated (as control) and treated with 100 µg/mL of PCAgNCs, were dropped on the glass slide and then incubated at 37 °C for 8 h. The samples were fixed with 2.5% glutaraldehyde in PBS and washed with a deionized water, and the suspension liquid resulting in a thin layer of bacteria was evaporated. The tested samples were examined by scanning electron microscopy (SEM; Tiny-SEM, Technex Lab Co. Ltd., Tokyo, Japan) with AuPd sputter coating by MSP-S1 magnetron sputter (Vacuum Device Inc., Ibaraki, Japan).

The released silver ion (Ag+) measurement. The released Ag+ was determined using HI96737 portable photometer (Hanna Instruments, Inc., Rhode Island, USA). PC-AgNCs were dispersed in Milli-Q water to prepare the 100 µg/mL suspension. The amount of Ag+ released from PCAgNCs was monitored after incubation for 20 min, 1 day and 3 days at 37 °C.

ASSOCIATED CONTENT Supporting information The Supporting Information is available free of charge on the ACS Publications website at DOI:XXX. Figures showing DLS data of PC-AgNCs (Figure S1); TGA curve of PC-AgNCs (Figure S2). UV–Vis absorption spectra of PC-AgNCs (Figure S31); Released Ag+ ions from PC-AgNCs (Figure S4).

ACS Paragon Plus Environment

Page 19 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

AUTHOR INFORMATION Corresponding Author *E-mail:[email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT A part of this study was supported by JSPS KAKENHI 16H03185.

REFERENCES [1] Xu, D., Wang, Q., Yang, T., Cao, J., Lin, Q., Yuan, Z., and Li, L. (2016) Polyethyleneimine capped silver nanoclusters as efficient antibacterial agents, Int. J. Environ. Res. Public Healt 13, 334-344. [2] Dos Santos, C. A., Seckler, M. M., Ingle, A. P., Gupta, I., Galdiero, S., Galdiero, M., Gade, A., and Rai, M. (2014) Silver Nanoparticles: Therapeutical Uses, Toxicity, and Safety Issues, J. Pharm. Sci. 103, 1931-1944. [3] Gliga, A. R., Skoglund, S., Odnevall Wallinder, I., Fadeel, B., and Karlsson, H. L. (2014) Size-dependent cytotoxicity of silver nanoparticles in human lung cells: the role of cellular uptake, agglomeration and Ag release, Part Fibre Toxicol. 11, 11-11. [4] Ravishankar Rai, V., and Jamuna Bai, A. (2011) Nanoparticles and their potential application as antimicrobials, Sci. Microb. Pathog.: Commun. Curr. Res. Technol. Adv., 197-209. [5] Durán, N., Marcato, P. D., Conti, R. D., Alves, O. L., Costa, F., and Brocchi, M. (2010) Potential use of silver nanoparticles on pathogenic bacteria, their toxicity and possible mechanisms of action, J. Braz. Chem. Soc. 21, 949-959. [6] Guzman, M., Dille, J., and Godet, S. (2012) Synthesis and antibacterial activity of silver nanoparticles against gram-positive and gram-negative bacteria, Nanomedicine 8, 37-45. [7] El Badawy, A. M., Silva, R. G., Morris, B., Scheckel, K. G., Suidan, M. T., and Tolaymat, T. M. (2011) Surface charge-dependent toxicity of silver nanoparticles, Environ. Sci. Technol. 45, 283-287. [8] Taglietti, A., Diaz Fernandez, Y. A., Amato, E., Cucca, L., Dacarro, G., Grisoli, P., Necchi, V., Pallavicini, P., Pasotti, L., and Patrini, M. (2012) Antibacterial activity of glutathionecoated silver nanoparticles against gram positive and gram negative bacteria, Langmuir

ACS Paragon Plus Environment

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 22

28, 8140-8148. [9] Jose Ruben, M., Jose Luis, E., Alejandra, C., Katherine, H., Juan, B. K., Jose Tapia, R., and Miguel Jose, Y. (2005) The bactericidal effect of silver nanoparticles, Nanotechnology 16, 2346-2353. [10] Diez, I., and Ras, R. H. A. (2011) Fluorescent silver nanoclusters, Nanoscale 3, 1963-1970. [11] Hu, B., Cao, X., and Zhang, P. (2013) Selective colorimetric detection of glutathione based on quasi-stable gold nanoparticles assembly, New J. Chem. 37, 3853-3856. [12] Zhu, J., Zhang, L., Teng, Y., Lou, B., Jia, X., Gu, X., and Wang, E. (2015) G-quadruplex enhanced fluorescence of DNA-silver nanoclusters and their application in bioimaging, Nanoscale 7, 13224-13229. [13] Zhang, J. R., Wang, Z. L., Qu, F., Luo, H. Q., and Li, N. B. (2014) Polyethyleniminecapped silver nanoclusters as a fluorescence probe for highly sensitive detection of folic acid through a two-step electron-transfer process, J. Agric. Food. Chem. 62, 6592-6599. [14] Chen, F., Tu, J., Liang, C., Yang, B., Chen, C., Chen, X., and Cai, C. (2016) Fluorescent drug screening based on aggregation of DNA-templated silver nanoclusters, and its application to iridium (III) derived anticancer drugs, Microchim. Acta 183, 1571-1577. [15] Wang, J., Wang, X., Wu, S., Song, J., Zhao, Y., Ge, Y., and Meng, C. (2016) Fabrication of highly catalytic silver nanoclusters/graphene oxide nanocomposite as nanotag for sensitive electrochemical immunoassay, Anal. Chim. Acta 906, 80-88. [16] Shang, L., Dorlich, R. M., Trouillet, V., Bruns, M., and Nienhaus, G. U. (2012) Ultrasmall fluorescent silver nanoclusters: Protein adsorption and its effects on cellular responses, Nano Res. 5, 531-542. [17] Setyawati, M. I., Yuan, X., Xie, J., and Leong, D. T. (2014) The influence of lysosomal stability of silver nanomaterials on their toxicity to human cells, Biomaterials 35, 67076715. [18] Iwasaki, Y., and Ishihara, K. (2012) Cell membrane-inspired phospholipid polymers for developing medical devices with excellent biointerfaces, Sci. Technol. Adv. Mater. 13, 064101. [19] Sangsuwan, A., Kawasaki, H., and Iwasaki, Y. (2016) Thiolated-2-methacryloyloxyethyl phosphorylcholine protected silver nanoparticles as novel photo-induced cell-killing agents, Colloids Surf., B 140, 128-134. [20] Boca, S. C., Potara, M., Gabudean, A.-M., Juhem, A., Baldeck, P. L., and Astilean, S. (2011) Chitosan-coated triangular silver nanoparticles as a novel class of biocompatible, highly effective photothermal transducers for in vitro cancer cell therapy, Cancer Lett. 311, 131-140. [21] Guo, D., Zhu, L., Huang, Z., Zhou, H., Ge, Y., Ma, W., Wu, J., Zhang, X., Zhou, X., Zhang, Y., et al. (2013) Anti-leukemia activity of PVP-coated silver nanoparticles via generation of reactive oxygen species and release of silver ions, Biomaterials 34, 7884-7894. [22] Chakraborty, I., Erusappan, J., Govindarajan, A., Sugi, K. S., Udayabhaskararao, T., Ghosh, A., and Pradeep, T. (2014) Emergence of metallicity in silver clusters in the 150 atom regime: a study of differently sized silver clusters, Nanoscale 6, 8024-8031. [23] Xu, H., and Suslick, K. S. (2010) Water‐Soluble fluorescent silver nanoclusters, Adv. Mater. 22, 1078-1082. [24] Yen, H. J., Hsu, S. H., and Tsai, C. L. (2009) Cytotoxicity and immunological response of gold and silver nanoparticles of different sizes, Small (Weinheim an der Bergstrasse, Germany) 5, 1553-1561.

ACS Paragon Plus Environment

Page 21 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

[25] Goda, T., Goto, Y., and Ishihara, K. (2010) Cell-penetrating macromolecules: direct penetration of amphipathic phospholipid polymers across plasma membrane of living cells, Biomaterials 31, 2380-2387. [26] Huang, Z., Jiang, X., Guo, D., and Gu, N. (2011) Controllable synthesis and biomedical applications of silver nanomaterials, J. Nanosci. Nanotechnol. 11, 9395-9408. [27] Goy, R. C., Morais, S. T. B., and Assis, O. B. G. (2016) Evaluation of the antimicrobial activity of chitosan and its quaternized derivative on E. coli and S. aureus growth, Rev. Bras. 26, 122-127. [28] Javani, S., Lorca, R., Latorre, A., Flors, C., Cortajarena, A. L., and Somoza, Á. (2016) Antibacterial activity of DNA-stabilized silver nanoclusters tuned by oligonucleotide sequence, ACS Appl. Mater. Interfaces. 8, 10147-10154. [29] Radtsig, M., Koksharova, O., and Khmel, I. (2008) [Antibacterial effects of silver ions: effect on gram-negative bacteria growth and biofilm formation], Molekuliarnaia genetika, mikrobiologiia i virusologiia, 27-31. [30] Jiao, Z. H., Li, M., Feng, Y. X., Shi, J. C., Zhang, J., and Shao, B. (2014) Hormesis Effects of Silver Nanoparticles at Non-Cytotoxic Doses to Human Hepatoma Cells, Plos One 9, 12. [31] Sambale, F., Wagner, S., Stahl, F., Khaydarov, R. R., Scheper, T., and Bahnemann, D. (2015) Investigations of the Toxic Effect of Silver Nanoparticles on Mammalian Cell Lines, Journal of Nanomaterials, 9. [32] Conner, S. D., and Schmid, S. L. (2003) Regulated portals of entry into the cell, Nature 422, 37-44. [33] Goto, Y., Matsuno, R., Konno, T., Takai, M., and Ishihara, K. (2008) Artificial cell membrane-covered nanoparticles embedding quantum dots as stable and highly sensitive fluorescence bioimaging probes, Biomacromolecules 9, 3252-3257. [34] Iwata, R., Suk-In, P., Hoven, V. P., Takahara, A., Akiyoshi, K., and Iwasaki, Y. (2004) Control of nanobiointerfaces generated from well-defined biomimetic polymer brushes for protein and cell manipulations, Biomacromolecules 5, 2308-2314. [35] Epand, R. M., and Epand, R. F. (2009) Lipid domains in bacterial membranes and the action of antimicrobial agents, Biochim. Biophys. Acta, Rev. Biomembr. 1788, 289-294. [36] Frimmel, F. H., and Niessner, R. (2010) Nanoparticles in the water cycle : properties, analysis and environmental relevance, Springer, Heidelberg; New York. [37] Goda, T., Tabata, M., Sanjoh, M., Uchimura, M., Iwasaki, Y., and Miyahara, Y. (2013) Thiolated 2-methacryloyloxyethyl phosphorylcholine for an antifouling biosensor platform, Chem. Commun. 49, 8683-8685. [38] Kitagawa, M., Matsumura, Y., and Tsuchido, T. (2000) Small heat shock proteins, IbpA and IbpB, are involved in resistances to heat and superoxide stresses in Escherichia coli, FEMS Microbiol. Lett. 184, 165-171. [39] Hiramatsu, K., Aritaka, N., Hanaki, H., Kawasaki, S., Hosoda, Y., Hori, S., Fukuchi, Y., and Kobayashi, I. (1997) Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin, Lancet (London, England) 350, 16701673. [40] Sondi, I., and Salopek-Sondi, B. (2004) Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria, J. Colloid Interface Sci. 275, 177182.

ACS Paragon Plus Environment

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Paragon Plus Environment

Page 22 of 22