CS

Subscriber access provided by UNIV OF LOUISIANA

Applications of Polymer, Composite, and Coating Materials

Rapid and Highly Effective Non-invasive Disinfection by Hybrid Ag/CS@MnO2 Nanosheets Using Near-Infrared Light Xiuhua Wang, Kun Su, Lei Tan, Xiangmei Liu, Zhenduo Cui, Doudou Jing, Xianjin Yang, Yanqin Liang, Zhaoyang Li, Shengli Zhu, Kelvin Wai Kwok Yeung, Dong Zheng, and Shuilin Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 1, 2019

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 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 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.

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 49 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 Applied Materials & Interfaces

Rapid and Highly Effective Non-invasive Disinfection by Hybrid Ag/CS@MnO2 Nanosheets Using Near-Infrared Light Xiuhua Wang a, Kun Su a, Lei Tan a, Xiangmei Liu a*, Zhenduo Cui b, Doudou Jing c, Xianjin Yang b, Yanqin Liang b, Zhaoyang Li b, Shengli Zhu b, Kelvin Wai Kwok Yeung d, Dong Zheng c, Shuilin Wu a,b* a

Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei Key Laboratory of Polymer Materials, School of Materials Science & Engineering, Hubei University, Wuhan 430062, China b

School of Materials Science & Engineering, the Key Laboratory of Advanced

Ceramics and Machining Technology by the Ministry of Education of China, Tianjin University, Tianjin 300072, China c

Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan 430022, China dDepartment

of Orthopaedics & Traumatology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China * To whom correspondence should be addressed:

E-mail: [email protected] (X.M. Liu);[email protected]; [email protected] (SL Wu) KEYWORDS: bacterial infection; MnO2 nanosheets; antibacterial; photothermal therapy; near infrared light 1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 The bacterial infection on the surface of medical apparatus and instruments as well as artificial implants is threatening the human health greatly. Antibiotics and traditional bacterial-killing agents even silver nanoparticles can induce bacterial resistance during long-term interaction with bacteria. Hence, rapid surface sterilization and prevention of bacterial infection in a long term are urgent for biomedical devices especially for artificial implant materials. Herein, a hybridized chitosan (CS), silver nanoparticles (AgNPs) and MnO2 nanosheets coating was designed on the surface of titanium plates, which can ensure the implants with rapid and highly effective antibacterial efficacy of 99.00% against Staphylococcus aureus (S. aureus) and 99.25% against Escherichia coli (E. coli) within 20 min of 808 nm near-infrared light (NIR) irradiation. The exogenous NIR irradiation can trigger the MnO2 nanosheets to produce enough hyperthermia within 10 minutes, which can combine with low concentration of pre-released Ag+ from the coating to achieve superior antimicrobial efficacy through synergistic effects. As a contrast, either pre-released Ag ions or photothermal effect alone can achieve much lower antibacterial efficiency under the same concentration, i.e., 24.00% and 30.01% for the former while 30.00% and 42.54% for the later toward S. aureus and E. coli, severally. The possible cytotoxicity of coatings could be eliminated owing to the low concentration of AgNPs and chitosan encapsulation. Thus, the novel bifunctional coating Ag/CS@MnO2 can exhibit great potential in deep site disinfection of Ti implants through the synergy of pre-released Ag ions and photothermal effect within a short time. 2 ACS Paragon Plus Environment

Page 2 of 49

Page 3 of 49 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


1. INTRODUCTION Bacterial infection, resulting in not only implant failure, but also morbidity, mortality, and complication, is a severe side effect after surgery.1 Generally, the biomaterials used for medical apparatus and artificial implants do not possess inherent antibacterial ability. At present, the commonly used method is to employ antibiotics to treat implant-associated infections in clinic. However, overuse of antibiotics can induce drug-resistance2 and even the occurrence of super-bacteria.3 The developing strategies include antibacterial substrates and antibacterial surface. The former is to add small amount of antibacterial metals into the substrate to form alloys during melting process, which can kill bacteria by leaching metallic ions such as Ag,4 and Cu.5,6 The latter is to build antibacterial coatings on the surface, including coatings with antibiotics loading,7

inorganic

bactericide

doping,8,9

organic

antibacterial10

and

adhesion-resistant materials intermingling.11 Unfortunately, these strategies can also induce some adverse effects such as bacterial resistance to loaded antibiotics,12,13 cell and tissue toxicity of released metallic ions or exfoliated nanoparticles.14-16 Ag as a nonspecific bactericidal agent, has a strong impact on a broad spectrum of bacterial and fungal species including strains with antibiotics resistance,17 which is due to the following two factors. One is that the permeability of the bacteria membrane can be increased by the accumulation of Ag nanoparticles (AgNPs) on the bacterial membrane, thus leading to cell death.18 The other is that free Ag ions can destroy bacterial metabolism.19,20 However, it also can induce bacterial resistance during long-term interaction with bacteria,21 because it often takes long time (several hours or more) to kill bacteria at a much higher concentration. In addition, Ag ions in 3 ACS Paragon Plus Environment


high concentrations can result in cells and tissue toxicity.22,23 In view of this, new surface strategies are quite important to assist Ag ions with a lower concentration to achieve a higher antibacterial efficacy within a short time, and meantime to avoid bacterial resistance caused by long-term interaction and toxicity induced by higher concentration of Ag ions. Recently, with the assistance of 808 nm light irradiation, photothermal therapy (PTT) is becoming a hopeful strategy for noninvasive therapy,24 especially for target cancer therapy,25,26 because of the fact that 700–1100 nm near infrared (NIR) light has deeper penetration ability to tissues, making it suitable for biological applications.27 Additionally, PTT based on NIR light could reach an effective treatment since NIR light can focus on a targeted area. And NIR light triggered PTT typically requires temperatures of 50 oC or higher to induce proteins disruption and bacteria death, but the high temperature or a long-time irradiation may cause scald and thermal injury to the tissues nearby.28,29 In addition, a lower temperature (e.g., 43 oC) strategy cannot reach a high antibacterial effect in a short time.30 So other strategies of co-ordination are needed. Manganese dioxide (MnO2) nanosheet, a representative of ultrathin layered semiconductor, has been attractive in applications of biology like fluorescent sensing, pH regulation and O2 generation,31-35 and it possesses excellent biocompatibility due to its degradability without unexpected adverse effects in vivo.36-38 However, as far as we know, few studies have reported the antibacterial applications of MnO2 nanosheet as a photothermal agent.

4 ACS Paragon Plus Environment

Page 4 of 49

Page 5 of 49 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


Scheme 1. Schematic drawing exhibiting the synergistic behaviors in the bacteria-killing

processes

of

Ag/CS@MnO2-Ti

through

pre-released

low

concerntration Ag+ and photothermal effects of MnO2 in the coating under NIR irradiation.

In the present work, a hybrid surface coating of 2D MnO2 nanosheets and chitosan (CS) modified Ag nanoparticles was constructed on metallic Ti plates (labeled as Ag/CS@MnO2-Ti) using a concisely hydrothermal synthesis method and subsequent electrostatic binding. As illustrated in Scheme 1, after a low concentration of Ag+ was locally released from this coating, the followed local hyperthermia produced from NIR light irradiated MnO2 nanosheets would make those bacteria inactive and increase the permeability of the bacterial membrane, which made them more easily 5 ACS Paragon Plus Environment


suffer from the attack of pre-existed small amount of released Ag+, finally causing the damage of bacterial membrane, proteins leakage and the decrease of ATP level. Meanwhile, during this course, MnO2 nanosheets could react with GSH at the broken membrane in acidic environment, in which MnO2 would be degraded into Mn2+ while GSH could be oxidized into glutathione disulfide (GSSG). This course could be accelerated with the assistance of hyperthermia according to our results. Since the oxidation of GSH in bacteria could induce the disorder of the intrinsic balance and consequently accelerate the bacterial death more easily. Hence, it could be speculated that the synergistic action of hyperthermia, Ag+ and MnO2 induced GSH oxidation could endow the hybrid coating with a superior antibacterial efficacy in a short time.

2. EXPERIMENTAL PROCEDURE 2.1 Construction of the hybrid system Biomedical Ti plates of 6 mm in diameter and 2.5 mm in thickness were used as the starting materials, which were ground and washed refers to our previous work.39 MnO2 nanosheets were hydrothermally prepared on Ti plates. Briefly, 0.225 g KMnO4 and 0.5 mL HCl with a concentration of 37% were instilled into 40 mL ultrapure water with 30 min vigorous stirring, after wholly mixed, the mixtures were poured into a 100 mL autoclave, and subsequently these samples were treated in an autoclave with the above solution for 12 h at 100 oC. Afterwards, these plates were washed 3 times in deionized water and alcohol, then MnO2 modified Ti plates were harvested after drying at 60 °C overnight (labeled as MnO2-Ti). 6 ACS Paragon Plus Environment

Page 6 of 49

Page 7 of 49 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


CS/Ag composites with different contents of AgNPs were synthesized as the following process. 2 mL of freshly prepared AgNO3 solution with different concentrations (1, 3 and 5 mg/mL) was instilled into 30 mL with 0.2% (w/v) chitosan (dissolved in 0.1% acetic acid solution) in a conical flask, respectively, and then the flask was heated to 95 oC with a constant stirring. Afterwards, different volumes of 0.3 M NaOH solution were added dropwise until the colloidal solution was turned from colorless to yellow, indicating the formation of AgNPs. After 10 min, the reaction stopped and the sediments were centrifuged and washed with sterile distilled water and alcohol. Then these precipitates were dried and collected to prepare three kinds of concentrations (labeled as 1-Ag/CS, 3-Ag/CS and 5-Ag/CS). Then these solutions were stored at -4 oC for further use. The prepared MnO2-Ti were immersed in the above Ag/CS solution in a vacuum environment for 24 h at 25 oC to obtain Ag/CS@MnO2-Ti through electrostatic adsorption. The harvested products were subsequently dried under vacuum at 25 oC and labeled as 1-Ag/CS@MnO2-Ti, 3-Ag/CS@MnO2-Ti and 5-Ag/CS@MnO2-Ti. For comparison, pure CS modified MnO2-Ti was labeled as CS@ MnO2-Ti. 2.2 Characterization The formation of AgNPs in CS was examined using UV-vis spectrum (SpectraMax I3, Molecular Devices), transmission electron microscopy (TEM, Tecnai G20, FEI, USA) and FTIR (NICOLET iS10). The morphologies of MnO2, CS@MnO2 or Ag/CS@MnO2 modified Ti plates were measured by SEM (JSM-6510LV, JEOL) and high-resolution TEM (FEI, TF20, USA). The existence of 7 ACS Paragon Plus Environment


MnO2, CS@MnO2 or Ag/CS@MnO2 on the substrate was detected using a SEM equipped energy-dispersive X-ray spectroscopy (EDS, JSM-6510LV) and X-ray diffraction (XRD, D8A25, Bruker, Germany). The zeta potential of modified plates was examined by Malvern zetasizer ZSTM (Malvern Instruments, UK). The compositional analysis was conducted using XPS. The force-displacement curves of different samples were measured using a nanoscratch tester produced by Hysitron in USA. 2.3 Photothermal effect of MnO2 nanosheets in vitro To determine the thermal conversion efficiency of MnO2 hybrid nanosheets on Ti plates, the nanosheets on the Ti plates were scraped off and collected, and then the powders were dissolved in deionized water homogeneously using ultrasonic dispersion (the concentration was fixed to 200 µg/mL). Then the solutions were irradiated under 0.5 W/cm2 NIR light of 808 nm. The records of real-time temperature were conducted using an infrared thermal imaging instrument (FLIR, E50) with a time interval of 1 min, and the photothermal conversion efficiency was calculated on the basis of the reported ways.40,41 The photothermal tests of MnO2-Ti, CS@MnO2-Ti and 5-Ag/CS@MnO2-Ti were conducted with same process for MnO2 nanosheets. Briefly, different samples underwent treatments of immersing in 200 μL PBS solution in 96-well plates and following 20 min exposure to light. Surface temperatures of plates were monitored using an infrared thermal imaging device with a time interval of 1 min. To obtain the


Page 8 of 49

Page 9 of 49 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


heating/cooling curves, the real-time temperature of 5-Ag/CS@MnO2-Ti was recorded in three cycles with 20-min light on and 10-min light off. 2.4 Ag+ release For determining the leaching behaviors of Ag+, 3 pieces of modified Ti plates were placed in 4 ml PBS (pH 7.4, 37 oC), then 2 mL of solution was taken out at different setting time: 1, 2, 3, 4, 5, 7, 10, 15, 20, 25 and 30 days. Then 2 mL of PBS were replenished after each time. The released Ag+ concentrations in PBS were detected using inductive coupled plasma atomic emission spectrometry (ICP-AES, Optimal 8000, PE, USA). Those pre-released Ag ions from different samples with 3 h immersion in PBS were determined using the same process. 2.5 In vivo antibacterial tests 2.5.1 Spread plate The antibacterial activity was investigated through commonly used spread plate method. As representative bacteria, Staphylococcus aureus (S. aureus, ATCC 25923) and Escherichia coli (E. coli, ATCC 8099) were used, which were cultured separately in the sterile Luria-Bertain (LB) media at 37 oC. The bacterial suspension was diluted with LB media until the concentration was 107 CFU/mL. Different samples (Pure Ti, MnO2-Ti,

CS@MnO2-Ti,

1-Ag/CS@MnO2-Ti,

3-Ag/CS@MnO2-Ti

and

5-Ag/CS@MnO2-Ti) were placed in 96-well plates, then 200 µL diluted suspension were added in each well. The samples were incubated in the media for 3 h to get a lower content of Ag+. Afterwards, the samples were illuminated under 0.5 W/cm2 808 nm NIR light for 20 min. While the surface temperature of MnO2-Ti, CS@MnO2-Ti 9 ACS Paragon Plus Environment


Page 10 of 49

and 1-Ag/CS@MnO2-Ti, 3-Ag/CS@MnO2-Ti and 5-Ag/CS@MnO2-Ti were raised to 50 oC within 7 min and kept for 13 min no more than 55 oC. Then the suspension of S. aureus and E. coli was diluted 200 and 100 times, respectively. 10 µL diluted suspension was spread uniformly on the agar medium with subsequent incubation of 24 h at 37 oC, and then the photographs of different agar plates were taken. The long-term antibacterial test in the dark was just treated with the incubation for 24 h at 37 oC only. The antibacterial efficiency of each plate was quantified by the count of colonies on the plates using the following formula: Bactericidal Ratio (%) =

CFUcontrol − CFUsample × 100% CFUcontrol

.

2.5.2 Bacterial morphology evluation The morphology and adhesion behavior of S. aureus and E. coli on the Ti plates were investigated by SEM and TEM observation. The bacteria on the Ti disks after treated by the above procedures underwent a similar process refers to the previous work.39 Then the Ti plates were observed by SEM the get the bacterial morphology, and the thin section containing the bacteria in TEM observation was used to determining the inner structure of the bacteria. 2.5.3 Live/dead evaluation After co-culturing with the samples and light irradiation, the above bacterial suspensions were discarded and the modified Ti plates were rinsed 2 times using PBS to wipe off LB medium. The staining of live/dead bacteria were carried out for 20 min in 5 × 10-6 M SYTO9 and 30 × 10-6 M PI at room temperature. After washing 2 times 10 ACS Paragon Plus Environment

Page 11 of 49 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


with PBS and drying at room temperature, the Ti plates were visualized using an Olympus inverted fluorescence microscope (IX73) to obtain bacterial images from random positions on different samples. 2.6 Antimicrobial mechanism 2.6.1 Ellman’s assay GSH oxidation was measured by a previous method.42 Briefly, the samples (Pure Ti, MnO2-Ti,

CS@MnO2-Ti

and

Ag/CS@MnO2-Ti

(5-Ag/CS@MnO2-Ti))

were

immersed with 150 µL 0.8 mM GSH bicarbonate buffer solution in 96-well plate and kept for 20 min to reach the adsorption and desorption equilibrium. Then those plates were split into two groups, one was exposed under NIR light for 20 min (0.5 W/cm2), the other was in dark at room temperature. Next, 450 μL of Tris-HCl (pH 8.0) solution with the concentration of 50 mM and 100 μL of the above used bicarbonate buffer solution with 10 mM DTNB were instilled into the GSH solution after those samples were taken out of it. The solutions were further put in a rocking bed for 20 min to fully react, and then measured through microplate reader at 410 nm. The GSH losing can be conducted according to the following equation: LOSS GSH (%) =

ODcontrol − ODsample × 100% ODcontrol

Where OD control and OD sample were the absorbance of negative control and samples at 410 nm, respectively. 2.6.2 Protein leakage Quantitative measurement of protein leakage from bacteria was based on a previous method.43 Briefly, 5×107 CFU mL-1 of S. aureus and E. coli were incubated with pure 11 ACS Paragon Plus Environment


Ti, MnO2-Ti, CS@MnO2-Ti and Ag/CS@MnO2-Ti (5-Ag/CS@MnO2-Ti), the samples were illuminated under 808 nm light sources with the same parameter as the above. The bacterial solution with pure Ti was the control group. Then the solutions were centrifuged for 10 min at 4 oC with the speed of 5,000 rpm. Afterwards, the supernatant liquids were transferred to a 96-well plate and the protein leakage concentrations were measured using BCA Protein Assay Kit (cat# P0010, Beyotime) on a microplate reader. 2.6.3 Adenosine triphosphate (ATP) test ATP is an energy molecule for the metabolic activity of bacteria.44 To evaluate the ATP level, 5×107 CFU mL-1 of S. aureus and E. coli were incubated in 96-well plates after the addition of different samples (pure Ti, MnO2-Ti, CS@MnO2-Ti and 5-Ag/CS@MnO2-Ti) and then exposed to NIR light sources (0.5 W/cm2, 20 min). The bacterial solution with pure Ti was the control group. And the solution was centrifuged for 10 min at 4 °C with the speed of 5,000 rpm and suspended in 300 µL of lysis buffer. The lysis buffer solution was transferred to an ultrasonic cell disruptor (vcx500; Sonics) and operated at a power of 30% with 3 s on/5 s off setting for 5 min in an ice-water bath. Then the upper-liquid was transferred to a 96-well plate and the ATP activities of all samples were monitored on a microplate reader. 2.7 In vitro cytotoxicity studies 2.7.1 MTT assay The cytotoxicity of cells on modified plates was assessed using 3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) test using NIH-3T3 12 ACS Paragon Plus Environment

Page 12 of 49

Page 13 of 49 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


cells. Those sterilized plates were placed in 96-well plates with 200 µL cell suspensions (104 cells/mL). After incubating for different times (1, 3 and 7 days), the original media was removed, and the samples were then kept for 4 h at 37 oC after the addition of 200 µL MTT solution (0.5 mg/mL) to make the ianthinus precipitate visible. After that, adding 200 µL DMSO to the wells after removing the MTT solution, to completely dissolve the crystals, and the wells were vibrated for 15 min. Finally, the absorption of the reacted medium at 490 nm was detected on a microplate reader after taking out the samples. The ratios of the experimental group and the control group were calculated to obtain the cell viability. The optical processing of MTT assay was conducted similar to the above procedure except for NIR irradiation (0.5 W/cm2) at the beginning in each culturing period (1, 3 and 7 days). The cells were attached fully on the samples after eight-hour culturing. Then each sample was treated with 808 nm light irradiation for 20 min, and in the meantime, the real-time temperature of photothermal effect was controlled no more than 55 oC, and then treated with MTT after culturing. The culture medium was refreshed every 2 days. 2.7.2 Adhesion and spreading assay To evaluate the cell adhesion, 200 mL diluted cell suspension (1×104 cells/mL) and different coatings modified Ti plates were co-cultured in a 96-well plate. After incubation with the same process to MTT assay at 37 oC in a 5% carbon dioxide incubator, the cells attached on the modified Ti plates after incubations of 1 and 3days were washed 2 times with PBS and dyed with FITC-Phalloidin (YiSen, China) for a time

duration

of

30

min,

then

the

plates

were


also

incubated

with


4,6-diamidino-2-phenylindole dihydrochloride (DAPI, YiSen, China) for 30 s in the absence of light. After washing with PBS for 2 times and drying, the cell morphologies were observed by the inverted fluorescence microscope. 2.8 In vivo antibacterial evaluation 2.8.1 Subcutaneous animal model The specific pathogen-free SD male rats with weight of 180 g obtained from Hubei Provincial Centers for Disease Prevention & Control were raised in isolation for acclimatization, after 7-days detection to build the subcutaneous infection model, the experimental procedure was conducted which was approved by Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. All rats were kept and conducted in following the Animal Management Rules of the Ministry of Health of the People’s Republic of China and the Guidelines for the Care and Use of Laboratory Animals of China. After grouping all acclimatized SD male rats randomly to two groups as pure Ti and Ag/CS@MnO2-Ti (5-Ag/CS@MnO2-Ti), and each group had 4 rats, the samples together with 20 µL of S. aureus suspension with the concentration of 1 × 107 CFU/mL were implanted in the subcutaneous tissue for further building of subcutaneous infection model. After culturing for 3 h to obtain the released Ag+, rats in a half of each group were irradiated with 0.5 W/cm2 NIR light of 808 nm (20 min). Then half of the implants were removed and 50 µL LB media was injected. After fully washing the surgical sites, 10 µL tissue fluids were extracted. The numbers of 14 ACS Paragon Plus Environment

Page 14 of 49

Page 15 of 49 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


bacteria in the tissue fluids were quantified by diluting the tissue fluids 400 times with LB media and spreading 20 µL onto agar plates. The rest of rats were bred in the same condition for 3 days, and then handled with the same spread plate methods as the above. 2.8.2 Histological analysis The tissues surrounding the implants after 3-days implantation extracting from each rat were fixed for 24 h in 4% formalin. The tissues underwent routine histological analysis and were cut into lateral sections with thickness of 4 µm. Then the sections were conducted with hematoxylin-eosin (H&E) and Giemsa staining to assess the bacterial contamination, and after that the viscera gathered from the rats were used to make histological section to observe the biological impacts on viscera. 2.9 Statistics In order to ensure the scientificity and accuracy of the experimental datas and results, the statistical significance against all the experiments data was evaluated and analyzed by a previous way.42

3. RESULTS AND DISCUSSION 3.1 Characterization of Ag/CS@MnO2-Ti The synthesis process of Ag/CS@MnO2-Ti was schematically illustrated in Figure 1A. During the synthesis of AgNPs-chitosan (Ag/CS) nano-composites, chitosan was utilized as a reducing and stabilizing agent to form AgNPs under a weakly alkaline condition, and the details were described in experimental procedure 2.1. UV-vis 15 ACS Paragon Plus Environment


spectra obtained from prepared CS and Ag/CS solutions are shown in Figure S1A. Obviously, pure CS solution has no absorption peak while the absorption intensity at 417 nm increases gradually along with the increasing of Ag content, and in addition, the color of solution turns to be yellow gradually (Figure S1B), indicating the formation of AgNPs. The further FT-IR determination (Figure S2) shows the peaks at 1090 cm-1 (C-O-C), 1370 cm-1 (asymmetrical C-H bending of CH2 group), 1640 cm-1 (C=O stretching), 2910 cm-1 (C-H stretching), and 3459 cm-1 (-OH and -NH2), indicating for the existence of CS.45 The dispersibility of Ag/CS nanocomposites is shown in Figure S3A, the size of AgNPs nanoparticles was ranging from 10 to 25 nm without aggregation. In addition, the selected area electron diffraction pattern (SAED) also turn out the formation of polycrystalline AgNPs (Figure S3B).


Page 16 of 49

Page 17 of 49 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 1. (A) Schematic drawing exhibiting the synthetic route of Ag/CS@MnO2-Ti. Characterization of MnO2-Ti: (B) Surface morphology and (C) cross-sectional images of MnO2-Ti, (D-F) HR-TEM images of scratched MnO2 nanosheets. Characterization of Ag/CS@MnO2-Ti: (G) Surface morphology and (H) cross-sectional images of Ag/CS@MnO2-Ti, (I-K) HR-TEM images of scratched Ag/CS@MnO2.

As shown in Figure 1B, after hydrothermal treatment in acidic KMnO4 solution, a regular and compact film composed of numerous nanosheets with a thickness about 50 nm (insert image in Figure 1B) appears on the surface of Ti. The prepared MnO2 nanofilm is 404 nm in thickness approximately (Figure 1C). TEM obtained from the 17 ACS Paragon Plus Environment


scratched film also reveals the nanosheet-liked structure of prepared MnO2 (Figure S4A). The high-resolution TEM images (Figure 1D to Figure 1F) discloses that the distances of lattice fringe of nanosheets with different orientation are 0.49 and 0.71 nm, respectively, which corresponds to the one of MnO2 planes according to a previous XRD pattern

46,

suggesting the successful preparation of MnO2 nanosheets

on the surface of Ti. The followed immersion in CS solution makes the film become smooth and more compact. As shown in Figure S5A, after immersion in CS, the nanosheet-like structure can still be observed (insert image in Figure S5A), and thickness is about 436 nm. As a contrast, after immersion in Ag/CS solution, the surface also exhibits smooth nanosheet-like structure (Figure 1G and insert image) with a thickness of 570 nm (Figure 1H) and some black nanoparticles ranging from the size of 5~20 nm were distributed on nanosheets (Figure S4B). The HR-TEM images obtained from the scratched hybrid film show that the lattice fringe distances from the black particle are 0.23 and 0.22 nm (Figure 1I to Figure 1K), which can be assigned to crystal planes of (111) and (200) of metallic Ag,47,48 indicating the existence of AgNPs in the coating. The EDS patterns obtained from the surface of prepared MnO2-Ti, CS@MnO2-Ti and Ag/CS@MnO2-Ti (Figure S6) also confirms existence of Mn, O, C, N, and Ag on the surface of the corresponding coatings. In addition, MnO2-Ti has the higher content of Mn, as the followed covering of CS or Ag/CS, the amount of Mn in these two coatings decreases with similar content, indicating the successful covering of CS or Ag/CS on MnO2-Ti, which is consistent with the results obtained from the cross-section images of the coatings. 18 ACS Paragon Plus Environment

Page 18 of 49

Page 19 of 49 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


According to the zeta potential (Figure S7), the surface potentials of MnO2, CS and Ag/CS are -23.5, 42.05 and 38.03 mV, respectively, indicating that the negatively charged MnO2 nanosheets could attach positively charged CS or Ag/CS composites by electrostatic adsorption.

Figure 2. (A) XRD spectra of MnO2, CS@MnO2 and Ag/CS@MnO2 composites. (B) XPS survey scan of MnO2-Ti, CS@MnO2-Ti and Ag/CS@MnO2-Ti. (C-E) High-resolution spectra of Mn 2p, Ag 3d and C 1s obtained from Ag/CS@MnO2-Ti.

The XRD and XPS were utilized to further determine the phase, and chemical compositions of prepared films. As shown in Figure 2A, peaks at 12.3o, 25.5o, 36.7o, and 66.2o were observed, corresponding to the crystal face of (001), (002), (111) and (311) of MnO2 nanosheets,49 and compared with MnO2-Ti, the characteristic peaks of AgNPs existed at 38.1o and 44.3o, which are assigned to the (111) and (200) diffractions of metallic Ag.50



XPS survey spectra shown in Figure 2B further prove the presence of Mn, O and Ti in MnO2-Ti, Mn, O, N and C in CS@MnO2-Ti, and Mn, O, N, C and Ag in Ag/CS@MnO2-Ti. According to the high-resolution spectra of Mn2p (Figure 2C), two typical peaks are located at the binding energy of 642.0 and 653.8 eV corresponding to the respective spin orbit of Mn (IV) 2p1/2 and Mn (IV) 2p3/2.51 And binding energy of 374.2 and 368.2 eV refer to the spin orbit peaks of Ag3d3/2 and Ag3d5/2 (Figure 2D), which are identical to the zero-valence state of Ag.52 The C 1s peak of Ag/CS@MnO2-Ti can be divided into four subpeaks at 284.5, 286.2, 288 and 292.7 eV, which were in accord with C-C/C-H, C-O, C=O and C-N, respectively (Figure 2E), indicating the characteristic peaks of chitosan. These results further confirm the successful preparation of three types of coatings on Ti plates. MnO2 nanosheets in-situ grew on the surface of pure Ti in the procedure of a hydrothermal reaction, in which the hydroxy formed on Ti during this course can provide a strong chemical bonding with MnO2 nanosheets layer, and the bonding strength was evaluated nanoscratch tests. As shown in Figure S8A and Figure S8B, the average bonding strength of the two kinds of coatings (MnO2 and Ag/CS@MnO2) was about 2500 μN, indicating that the coatings exhibited a strong bonding strength with substrate. The structural stability of the coatings was evaluated by XPS analysis. According to Figure S8C and Figure S8D, after immersion in PBS at 37 oC for 3 days, MnO2 coating showed the existence of Mn and O while Ag/CS@MnO2 displayed Mn, O, N and C signals, revealing that the electrostatic adsorption could provide strong bonding between the MnO2 nanosheets and CS/Ag to keep the structural stability of 20 ACS Paragon Plus Environment

Page 20 of 49

Page 21 of 49 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


Ag/CS@MnO2. It should be noted that no signal of Ag was detected on Ag/CS@MnO2, which was due to the 3-days release of Ag ions from the coating with a very lower concentration originally. 3.2 Photothermal properties The optical absorption of MnO2 nanosheets was measured by UV-Vis spectrum (Figure S9). The wavelength in 808 nm possesses high depth of the penetrating in skin and low-intensity absorbance by tissue, blood as well as water, which is known to be one of the most considerable NIR wavelengths for PTT.27 And MnO2 nanosheets appeared a heavy black color on Ti (insert image in Figure S9). The photothermal conversion (PTC) efficiency was measured and calculated in Figure 3A. It can be calculated that MnO2 nanosheets possess a high PTC of 30.79%. The temperature evolution curves were shown in Figure 3B. After 20 min irradiation by 808 nm NIR light, the temperature of pure Ti was raised to 32.5 oC barely, while it was 52.8, 51.6 and 55.4 oC for MnO2-Ti, CS@MnO2-Ti and Ag/CS@MnO2-Ti. CS@MnO2-Ti exhibited a little lower temperature than MnO2-Ti, which was possibly ascribed to the cover of CS on MnO2, hindering the light absorption for MnO2 slightly. However, Ag/CS@MnO2-Ti showed a slightly higher temperature than MnO2-Ti. This phenomenon was possibly caused by the surface plasmon resonance (SPR) of outer electrons of AgNPs in the Ag/CS@MnO2, which can inspire the photothermal effect of AgNPs,53,54 thus exhibiting a little higher temperature than pure MnO2. And Figure 3C illustrates the real-time photothermal images curves corresponding to Figure 3B, and it is obvious that NIR light can be absorbed and converted into heat via the 21 ACS Paragon Plus Environment


Page 22 of 49

photothermal effect of MnO2 nanosheets in the coating. Especially, according to the three heating and cooling cycles with no obvious temperature reductions (Figure 3D), the as prepared MnO2 nanosheets can exhibit good photothermal conversion stability. Therefore, it can be concluded that MnO2 nanosheets could be used to be a desirable photothermal agent for PTT.

Figure 3. (A) Photothermal curves of dispersed MnO2 nanosheets suspension (200 μg/mL) under 808 nm light irradiation at 0.5 W/cm2 and then shut off the light until the temperature reached a steady state, and the linear time results from the cooling stage (1200 s) relative to negative natural logarithm about driving-force temperature was applied in the heat transfer procedure from the system to obtain the time constant (τs =247.5 s). (B) The curves of temperature vs irradiation time of different samples. (C)

Real-time

photothermal

images

of

MnO2-Ti;


CS@MnO2-Ti

and

Page 23 of 49 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


Ag/CS@MnO2-Ti. (D) The heating/cooling profiles of Ag/CS@MnO2-Ti with 3 cycles under NIR irradiation (0.5 W/cm2).

3.3 Antibacterial activity in vitro The antibacterial activity of the samples toward S. aureus and E. coli was evaluated by spread plate technique, live/dead bacterial staining, bacterial section and bacterial morphologies, and related results are exhibited in Figure 4 and Figure S10, respectively. Obviously, after culturing for 3 h in the dark, all modified samples such as CS@MnO2-Ti and Ag/CS@MnO2-Ti show a slight decrease of bacterial colonies on the surface (Figure 4B and Figure S10B). The little antibacterial efficiency of MnO2-Ti is ascribed to its negative potential (Figure S7) to resist the adherence of bacteria with negative potential possibly. Although 5-Ag/CS@MnO2-Ti exhibits slightly higher antibacterial efficacies of 24% against S. aureus (Figure 4B) and 30.10% against E. coli (Figure S10B), this lower bacteria-killing efficacy cannot prevent the bacterial infection due to its lower Ag+ concentration of 79.59 μg/L released from coatings within 3 hours (Table S1). When handled with 20 min of 808 nm light irradiation, similar trend can be observed for S. aureus as well as E. coli. Compared with pure Ti, with the effect of the photothermal effect of MnO2, CS@MnO2-Ti group also exhibited poor antibacterial efficiencies of 30.67% and 60.95% toward S. aureus and E. coli, respectively, as a result of the possible electrostatic adsorption.55,56 Besides, a small part of bacterial can be inactivated and killed by a local hyperthermia. As a result, both MnO2-Ti and CS@MnO2-Ti exhibit decreased bacterial colonies. 23 ACS Paragon Plus Environment


However, lower temperature photothermal action below 60 oC can only kill a small number of bacteria alone within a short time, and hyperthermia over 60 oC with a long irradiation time will scald the normal cells and tissues.57 This is the reason that under the light irradiation of 808 nm light for 20 min, MnO2-Ti can only exhibit lower antibacterial efficiency of 30% and 42.53% toward S. aureus and E. coli in respective result. The above results suggested that either low concentration of Ag+ or low-temperature photothermal effects could not kill the bacteria effectively alone within a short time. As a contrast, 1-Ag/CS@MnO2-Ti, 3-Ag/CS@MnO2-Ti and 5-Ag/CS@MnO2-Ti exhibited increased antibacterial efficacy after culturing for 3 h with the followed NIR light irradiation for 20 min. In this case, group 5-Ag/CS@MnO2-Ti exhibited the highest antibacterial efficacy of 99.00% and 99.25% toward S. aureus and E. coli, respectively, indicating that the synergistic action could increase the antibacterial efficacy of coatings significantly during a short time even with a low concentration of Ag+ as well as low-temperature photothermal effect. Considering the fact that a high concentration of Ag may cause cytotoxicity or tissue toxicity,12 we chose 5-Ag/CS@MnO2-Ti for further study due to its higher in vitro antibacterial efficacy and relatively lower cytotoxicity shown in the Section 3.5. The bacterial viability on the surface of Ti plates was further assessed by Live/Dead fluorescence staining assay. And the red-fluorescent nucleic acid dye PI, which is able to penetrate damaged cell wall, was used to label dead bacteria. In comparison, the green-fluorescent nucleic acid dye SYTO9, which is able to penetrate damaged as well as intact cell membranes, was applied to be a label of live bacterial 24 ACS Paragon Plus Environment

Page 24 of 49

Page 25 of 49 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


cells.58 Figure 4C and Figure S10C show the fluorescence staining pictures of two types of bacteria cells on the surfaces of different modified Ti plates after culturing for 3 h. Obviously, both S. aureus and E. coli on all the surfaces were stained green without 808 nm light irradiation, namely, the bacteria were alive, indicating that all groups were suitable for bacterial growth without light. As a contrast, after 808 nm light irradiation, except for pure Ti group with green spots on whole surface, some red fluorescence spots (dead cells) together with partially distributed green spots can be observed on the surface of MnO2-Ti as well as CS@MnO2-Ti, showing that these two kinds of modified Ti plates have lower antibacterial efficacy, which is in accord with spread plate results both shown in Figure 4A and Figure S10A. In contrast, the groups of Ag/CS@MnO2-Ti exhibit almost all red spots on the surface, indicating that bacteria on the surface have been killed almost completely. The quantitative analysis of the fluorescence staining was calculated by the software of Image J (Figure S11). The result also showed that 5-Ag/CS@MnO2-Ti exhibited the lowest fluorescence area ratio of 0.11 and 0.18 toward S. aureus and E. coli, respectively, indicating the highest antibacterial efficacy of this group. Figure 4D and Figure S10D show the morphologies of two types of bacteria on the surfaces, respectively. Evidently, except for slight deformation of bacteria on 5-Ag/CS@MnO2-Ti, the bacteria exhibit regular and smooth shape with intact cell wall on the surface of pure Ti, MnO2-Ti and CS@MnO2-Ti after 3 h culturing, while the bacterial membranes exhibited different degrees of disruption like sagged, fracted, and deformed features (indicated by red arrows) in group MnO2-Ti, CS@MnO2-Ti 25 ACS Paragon Plus Environment


and 5-Ag/CS@MnO2-Ti with the irradiation of 808 nm light. These results are also in accord with the Live/Dead fluorescence staining test, i.e., pre-released Ag ions from the coatings and photothermal effect of MnO2 can exhibit synergistic bacteria-killing efficiency. The hyperthermia can assist Ag ions to cause membrane potential variation and intracellular oxidation more easily since AgNPs can play a role in delivering Ag+ to generate ROS and destroy the bacterial DNA laterly.59

Figure 4. Antibacterial characterization of S. aureus in vitro: (A) The spread plate results of different samples. (B) the corresponding antibacterial efficacy of different samples after culturing for 3 h and with the followed NIR irradiation for 20 min (808 nm, 0.5 W/cm2). The error bars indicated in the graph means ± SD (n = 3): *P < 0.05, **P < 0.01. (C) Fluorescence staining pictures of different samples, live bacteria were stained green and dead bacteria were stained red (scale bar: 20 µm). (D) the 26 ACS Paragon Plus Environment

Page 26 of 49

Page 27 of 49 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


morphologies of bacteria incubated with different samples after culturing for 3 h or co-treated with 808 nm light irradiation (0.5 W/cm2) for 20 min (scale bar = 1 μm). (E) TEM images and the corresponding EDS analysis of the bacterial intracellular sections of different samples (scar bar: 200 nm).

The bacterial section images shown in Figure 4E and Figure S10E further confirmed that pure Ti has no antibacterial ability even under 808 light irradiations, which was proven by the intact and thick bacterial membranes, while the bacterial membranes on 5-Ag/CS@MnO2-Ti are distorted or broken. In addition, the EDS spectrum shows that the Ag content in the broken bacterial cell is extremely low (0.03% and 0.44% in S. aureus and E. coli respectively), indicating that Ag was penetrated into the bacterial membrane. These results indicate that with the assistance of photothermal effects, Ag ions even with low concentration can easily enter into the inside of bacteria and destroy the membrane, intracellular proteins, and DNA and then resulting in the killing of bacteria. The long-term antibacterial efficacy of the samples can be evaluated after 24 h culturing without light irradiation. As shown in Figure S12A, the numbers of colonies on the surface of the groups pure Ti, MnO2-Ti and CS@MnO2-Ti do not decline obviously while 5-Ag/CS@MnO2-Ti has few bacterial colonies because the coating of 5-Ag/CS@MnO2 can release Ag ions after 24 h culturing to physically kill bacteria with an antibacterial efficacy of 95.11% and 96.01% toward S. aureus as well as E. coli, respectively (Figure S12B), which can be proven by the Ag ions release curves 27 ACS Paragon Plus Environment


shown in Figure S13. In addition, this gradual and slow release of Ag+ shown in Figure S13 can favor the prevention of bacterial infection in the long term. 3.4 Antibacterial mechanisms Glutathione (GSH), as a thiol-containing tripeptide, which exhibits a significant feature in the system of bacterial antioxidant defense, since it can indicate the oxidative stress in cells. Under oxidation conditions, glutathione disulfide (GSSG) was generated from GSH.60 And the MnO2 nanosheet can undergo a redox reaction with GSH to yield Mn2+ and GSSG.61 The existence of Mn2+ was detected by ICP-AES shown in Table S2. After light treatment, it is obvious that the group MnO2-Ti has the highest concentration of Mn2+ (1.402 mg/L), and with the coverage of CS composites, the other two groups exhibit some decrease, but the presence of Mn2+ still cannot be ignored. Besides, the losing of GSH is shown in Figure 5A. Without light irradiation, compared with pure Ti, MnO2-Ti exhibits a percentage of 46.88% toward GSH oxidation owning to the oxidizability of MnO2 under the existence of HCO3- buffer which is one of the most important physiological buffers. Pure Ti group almost has no oxidation effect to GSH, even under NIR irradiation. However, after the NIR irradiation for 20 min, the GSH losing level is about 79.76%, 81.96% and 75.99% for MnO2-Ti, CS@MnO2-Ti and Ag/CS@MnO2-Ti, respectively, and Figure 5B shown the corresponding visual images of color change after GSH treatment, The color of MnO2-Ti, CS@MnO2-Ti and Ag/CS@MnO2-Ti with NIR lights almost entirely faded, indicating that the oxidation of GSH was most serious, which is well corresponding to the results shown in Figure 5A. The generation of 28 ACS Paragon Plus Environment

Page 28 of 49

Page 29 of 49 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


GSSG from GSH was promoted by surface hyperthermia on Ag/CS@MnO2-Ti induced by 808 nm light irradiation. As a consequence, the synergistic action of hyperthermia and pre-released small amount of Ag+ made bacterial membrane broken, resulting in the leakage of internal substance. Thus, MnO2 nanosheets could also react with the GSH at the broken site, accelerating the disorder of the intrinsic balance and the death. Protein leakage from bacteria means the destruction of cell membrane, which is a main antibacterial mechanism of enhancing the permeability of membrane. According to Figure 5C and Figure S14A, after the irradiation of 808 nm light for 20 min with followed incubation of 3 h, Ag/CS@MnO2-Ti exhibits protein leakage of 1.7 and 1.9 times than pure Ti with light irradiation toward S. aureus and E. coli, respectively. The other groups do not show obvious changes, indicating that the bacterial membranes on the surface of 808 nm light irradiated Ag/CS@MnO2-Ti experienced the most severe breakdown in accordance with the bacterial sections in Figure 4E.



Figure 5. (A) the Ellman’s assay results showing the GSH losing histograms after incubation with samples under NIR treatments (0.5 W/cm2) for 20 min or not. (B) The corresponding color-changed pictures of different samples after GSH treatment. (C) Protein leakage (OD values at 562 nm) and (D) ATP level of the S. aureus suspensions after treating with different samples under light irradiation or not (808 nm, 0.5 W/cm2, 20 min) (*P < 0.05, **P < 0.01, ***P < 0.001).

As the major source of energy in bacterial intracellular reactions,62 ATP takes participation in the various processes in physiology and Pathology, and its level will drop after the cells suffer necrosis or apoptosis.63 Figure 5D and Figure S14B exhibited the ATP levels in the apoptosis of bacterial on the surface of samples caused by different treatments. After culturing for 3 h and the followed 808 nm light irradiation, the luminescence intensities of Ag/CS@MnO2-Ti among all groups were


Page 30 of 49

Page 31 of 49 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 lowest toward both S. aureus and E. coli, indicating best antibacterial efficacy of this kind of sample. 3.5 Cell viability in vitro To be applicable clinically, the artificial materials should possess both high antimicrobial capability and good biocompatibility. According to previous studies, Ag ions in high concentrations have cytotoxicity.64,65 And it is necessary to lower the cytotoxicity of Ag ions. The MTT test was applied to observe the cell viability. And Figure 6A shows the effects of light irradiation. Compared with pure Ti, even cell numbers decline for the modified samples after the cultivation for 1 day owning to the photothermal impact or Ag ions, but after culturing for 7 days, the cell viabilities were increased with cell viabilities higher than 70%. The results suggest that both Ag+ and hyperthermia of Ag/CS@MnO2-Ti can show some cytotoxicity in a certain time but these effects will decline in long-term cultivation. As shown in Figure 6B, compared with pure Ti, after culturing for 1, 3, and 7 days in the dark, MnO2-Ti and CS@MnO2-Ti exhibited cell viability in the range of 97-140% and 98-123%, respectively, indicating the excellent cytocompatibility of both MnO2 nanosheets and CS@MnO2 coatings. After AgNPs encapsulated, the decline could be observed, the group of 1-Ag/CS@MnO2-Ti, 3-Ag/CS@MnO2-Ti and 5-Ag/CS@MnO2-Ti exhibited cell viabilities of 67%, 64% and 57% after 1-day incubation. But as the incubation time prolonged, the cell reactivated in a largely part, even 5-Ag/CS@MnO2-Ti can have a lowest value of 80.52% after 7 days-incubation, disclosing that even the largest concentration of AgNPs in all experimental group possesses a relatively good cell 31 ACS Paragon Plus Environment


compatibility since CS can plays an important role of stabilize and reduce the impact on cell condition of Ag ions. The morphology and spreading activity of cells were determined by a normal method of FITC and DAPI staining, and Figure 6C and Figure 6D shown the fluorescence images of the F-actin and nuclei, after 1-day culturing. In comparison with pure Ti, 5-Ag/CS@MnO2-Ti exhibit different degrees of cell deceases with or without NIR irradiation, however, the number of cells on the 5-Ag/CS@MnO2-Ti recovers greatly after 3 days’ culturing. Besides, a superior adhesion with an extending morphology could be observed. The results of fluorescent images are also in agreement with MTT assay, indicating that MnO2 nanosheet and Ag/CS modified sample favors initial adhesion, spreading, and growth of cells with no obvious cytotoxicity.


Page 32 of 49

Page 33 of 49 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 6. (A) 808 nm (+) and (B) 808 nm (-) Cell viability of different samples (divided as the groups of Pure Ti, MnO2-Ti, CS@MnO2-Ti and Ag/CS@MnO2-Ti) for 1, 3, 7 days. (808 nm, 20 min, 0.5 W/cm2, *P < 0.05, **P < 0.01). Fluorescent images of cells on the surface of different samples (scar bar: 50 μm): (C) 808 nm (+) and (D) 808 nm (-).

3.6 In vivo evaluation The in vivo antibacterial efficacy of samples was evaluated by the subcutaneous infection model. As shown in Figure 7A and Figure 8A, after culturing for 3 h, compared with pure Ti, MnO2-Ti exhibited low antibacterial efficiency of 0.83% 33 ACS Paragon Plus Environment


Page 34 of 49

(Light-) and 43.89% (Light+) against S. aureus (Figure S15), and the antibacterial efficiency of 5-Ag/CS@MnO2-Ti was 29.23% (Light-) and 99.96% (Light+), respectively, suggesting the rapid and efficient antibacterial performance of 5-Ag/CS@MnO2-Ti in vivo under light irradiation. For the long-term antibacterial ability after 3 days, it was obvious that both Ag/CS@MnO2-Ti (Light+) and Ag/CS@MnO2-Ti (Light-) show antibacterial efficacy of 100% and 97.58%, respectively,

indicating

the

great

long-term

antibacterial

performance

of

Ag/CS@MnO2-Ti with or without light (Figure 7B and Figure 8B). These results confirmed that light illumination together with small amount of preleased Ag+ could endow the coating of Ag/CS@MnO2 with an immediate and superior antibacterial efficacy in vivo while the subsequent gradual release of Ag+ could provide killing of bacteria in the long-term. The former was attributed to the synergistic behavior of photothermal effect and Ag+ while the latter was attributed to long-term action of Ag+.

Figure 7. Representative images for the results of spread plates of S. aureus obtained from the subcutaneous infection mode under light irradiation or not (808 nm, 0.5 34 ACS Paragon Plus Environment

Page 35 of 49 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


W/cm2): (A) Cultured for 3 h and light for 20 min; (B) After culturing for 3 h and light for 20 min, the subcutaneous animal models were cultured for 3 days continuously.

The inflammation analysis of pure Ti and Ag/CS@MnO2-Ti treated rats was investigated through H&E staining. As shown in Figure 8C, it is obvious that large amount of inflammatory cells are observed in Pure Ti group (indicated by red arrows), while significantly reduced inflammatory cells are observed in the group of Ag/CS@MnO2-Ti. Meanwhile, the Giemsa staining was shown in Figure 8D, many S. aureus (indicated by red arrows) could be found from the tissues in pure Ti group. In comparison, the group of Ag/CS@MnO2-Ti with or without NIR irradiation exhibited none of bacteria, demonstrating that the Ag/CS@MnO2-Ti possessed rapid surface disinfection and prevention of bacterial infection in the longterm in vivo. Additionally, the histological analysis of the biological effects on viscera was examined by H&E staining (Figure S16). No visible organ damage or abnormity could be observed from the four groups after 3-day implantation, suggesting that the Ag/CS@MnO2-Ti exhibited great biocompability in vivo.



Figure 8. Antibacterial and histological assay in vivo: Antibacterial ratio histogram of S. aureus on different samples: (A) Culturing for 3 h and light for 20 min and (B) After culturing for 3 h and light for 20 min, and continuously cultured 3 days, the bacterial were determined by the number of colonies according to the results of spread plate method (**P < 0.01, ***P < 0.001). (C) and (D) the representative histological images of tissues by H&E and Giemsa staining (scar bar: 50 μm).

4. CONCLUSION In summary, Ag/CS composites have been successfully fabricated and loaded on MnO2 nanosheets modified Ti implants. The chitosan acted as a role of reduced and stabilized agent of AgNPs, and the low content of AgNPs encapsulated in chitosan composite possesses a slow-released property, which can reduce the cytotoxicity of Ag ions. When the bacteria were treated with photothermal effect of MnO2 nanosheets triggered by NIR light for 20 min, and the GSH could be reduced to GSSG, 36 ACS Paragon Plus Environment

Page 36 of 49

Page 37 of 49 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 bacteria are easily attacked. Thus, even the low-content Ag ions could induce bacterial membrane damage, protein leakage and ATP decreasing. As a consequence, compared with photothermal effect or Ag ions barely, the antibacterial efficacies of Ag/CS@MnO2-Ti toward S. aureus as well as E. coli were improved greatly. Besides, the biocompability was also enhanced greatly in vitro as well as in vivo. Since the good penetrating capacity of NIR light, this rapid in situ and long-term disinfection system shows great potential in the noninvasive treatment of implantation infection.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. The Supporting Information including Part I: Supplementary Figures S1-16, and Part II: Supplementary Table S1

AUTHOUR INFORMATION Corresponding Author To whom correspondence should be addressed: E-mail: [email protected] (X.M. Liu); [email protected]; [email protected] (SL Wu)

Notes The authors declare no competing financial interest.



Page 38 of 49

ACKNOWLEDGEMENTS This work is jointly supported by the National Natural Science Foundation of China, Nos. 51671081, 51871162, and 51801056, and the National Key Research and Development

Program

of

China

No.

2016YFC1100600

(sub-project

2016YFC1100604), and Natural Science Fund of Hubei Province, 2018CFA064.

REFERENCS (1) Wang, G. M.; Jin, W. H.; Qasim, A. M.; Gao, A.; Peng, X.; Li, W.; Feng, H. Q.; Chu, P. K. Antibacterial Effects of Titanium Embedded with Silver Nanoparticles Based on Electron-transfer-induced Reactive Oxygen Species. Biomaterials 2017, 124, 25-34. (2) Ralph, G. Overuse of Antibiotics for Acute Respiratory Illnesses and the Epidemic of Penicillin-resistant Streptococcus Pneumoniae. Drug Resist. Updates 1998, 1, 221-222. (3) Song, Y. Q.; Bai, J. L.; Zhang, R.; He, H. L.; Li, C.; Wang, J.; Li, S.; Peng, Y.; Ning, B. A.; Wang, M. L.; Gao, Z. X. Michael-Addition-Mediated Photonic Crystals Allow Pretreatment-Free and Label-Free Sensoring of Ciprofloxacin in Fish Farming Water. Anal. Chem. 2018, 90, 1388-1394. (4) Zheng, Y. F.; Zhang, B. B.; Wang, B. L.; Wang, Y. B.; Li, L.; Yang, Q. B.; Cui, L. S. Introduction of Antibacterial Function into Biomedical TiNi Shape Memory Alloy by the Addition of Element Ag. Acta Biomater. 2011, 7, 2758-2767. (5) Chai, H. W.; Guo, L.; Wang, X. T.; Fu, Y. P.; Guan, J. L.; Tan, L. L.; Ren, L.; 38 ACS Paragon Plus Environment

Page 39 of 49 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


Yang, K. Antibacterial Effect of 317L Stainless Steel Contained Copper in Prevention of Implant-related Infection in vitro and in vivo. J. Mater. Sci.: Mater. Med. 2011, 22, 2525-2535. (6) Finke, B. B.; Polak, M.; Hempel, F.; Rebl, H.; Zietz, C.; Stranak, V.; Lukowski, G.; Hippler, R.; Bader, R.; Nebe, J. B.; Weltmann, K. D.; Schro¨der, K. Antimicrobial Potential of Copper-Containing Titanium Surfaces Generated by Ion Implantation and Dual High Power Impulse Magnetron Sputtering. Adv. Eng. Mater. 2012, 14, 224-230. (7) Stigter, M.; Bezemer, J.; de Groot, K.; Layrolle, P. Incorporation of Different Antibiotics into Carbonated Hydroxyapatite Coatings on Titanium Implants, Release and Antibiotic Efficacy. J. Controlled Release 2004, 99, 127-137. (8) Mei, S. L.; Wang, H. Y.; Wang, W.; Tong, L. P.; Pan, H. B.; Ruan, C. S.; Ma, Q. L.; Liu, M. Y.; Yang, H. L.; Zhang, L.; Cheng, Y. C.; Zhang, Y. M.; Zhao, L. Z.; Chu, P. K. Antibacterial Effects and Biocompatibility of Titanium Surfaces with Graded Silver Incorporation in Titania Nanotubes. Biomaterials 2014, 35, 4255-4265. (9) Hu, H. J.; Zhang, W. J.; Qiao, Y. Q.; Jiang, X. Q.; Liu, X. Y.; Ding, C. X. Antibacterial Activity and Increased Bone Marrow Stem Cell Functions of Zn-incorporated TiO2 Coatings on Titanium. Acta Biomater. 2012, 8, 904-915. (10) Shi, Z. L.; Neoh, K. G.; Kang, E. T.; Poh, C.; Wang, W. Bacterial Adhesion and Osteoblast Function on Titanium with Surface-grafted Chitosan and Immobilized RGD Peptide. J. Biomed. Mater. Res. A 2008, 86, 865-872. (11) Zhang, F.; Zhang, Z. B.; Zhu, X. L.; Kang, E. T.; Neoh, K. G. 39 ACS Paragon Plus Environment


Page 40 of 49

Silk-functionalized Titanium Surfaces for Enhancing Osteoblast Functions and Reducing Bacterial Adhesion. Biomaterials 2008, 29, 4751-4759. (12) Chen, M. H.; Xie, S. Z.; Wei, J. J.; Song, X. J.; Ding, Z. H.; Li, X. H. Antibacterial

Micelles

with

Vancomycin-Mediated

Targeting

and

pH/Lipase-Triggered Release of Antibiotics. ACS Appl. Mater. Interfaces 2018, 10, 36814-36823. (13) Arciola, C. R.; Campoccia, D.; Speziale, P.; Montanaro, L.; Costerton, J. W. Biofilm Formation in Staphylococcus Implant Infections. A Review of Molecular Mechanisms and Implications for Biofilm-resistant Materials. Biomaterials 2012, 33, 5967-5982. (14) Rakow, A.; Schoon, J.; Dienelt, A.; John, T.; Textor, M.; Duda, G.; Perka, C.; Schulze, F.; Ode, A. Influence of Particulate and Dissociated Metal-on-metal Hip Endoprosthesis Wear on Mesenchymal Stromal Cells in vivo and in vitro. Biomaterials 2016, 98, 31-40. (15) Xie, C. M.; Lu, X.; Wang, K. F.; Meng, F. Z.; Jiang, O.; Zhang, H. P.; Zhi, W.; Fang, L. M. Silver Nanoparticles and Growth Factors Incorporated Hydroxyapatite Coatings on Metallic Implant Surfaces for Enhancement of Osteoinductivity and Antibacterial Properties. ACS Appl. Mater. Interfaces 2014, 6, 8580-8589. (16) Chen, W. Y.; Chang, H. Y.; Lu, J. K.; Huang, Y. C.; Harroun, S. G.; Tseng, Y. T.; Li, Y. J.; Huang, C. C.; Chang, H. T. Self-Assembly of Antimicrobial Peptides on Gold

Nanodots:

Against

Multidrug-Resistant

Bacteria

Application. Adv. Funct. Mater. 2015, 25, 7189-7199. 40 ACS Paragon Plus Environment

and

Wound-Healing

Page 41 of 49 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


(17) Cao, H. L.; Liu, X. Y.; Meng, F. H.; Chu, P. K. Biological Actions of Silver Nanoparticles Embedded in Titanium Controlled by Micro-galvanic Effects. Biomaterials 2011, 32, 693-705. (18) Sondi, I.; Salopek-Sondi, B. Silver Nanoparticles as Antimicrobial Agent: A Case Study on E. coli as a Model for Gram-negative Bacteria. J. Colloid Interface Sci. 2004, 275, 177-182. (19) Kim, J. S.; Kuk, E.; Yu, K. N.; Kim, J. H.; Park, S. J.; Lee, H. J.; Kim, S. H.; Park, Y. K.; Park, Y. H.; Hwang, C. Y.; Kim, Y. K.; Lee, Y. S.; Jeong, D. H.; Cho, M. H. Antimicrobial Effects of Silver Nanoparticles. Nanomedicine 2007, 3, 95-101. (20) Shrivastava, S.; Bera, T.; Roy, A.; Singh, G.; Ramachandrarao, P.; Dash, D. Characterization of Enhanced Antibacterial Effects of Novel Silver Nanoparticles. Nanotechnology 2007, 18, 225103. (21) Panacek, A.; Kvitek, L.; Smekalova, M.; Vecerova, R.; Kolar, M.; Roderova, M.; Dycka, F.; Sebela, M.; Prucek, R.; Tomanec, O.; Zboril, R. Bacterial Resistance to Silver Nanoparticles and How to Overcome it. Nat. Nanotechnol. 2018, 13, 65-71. (22) Xu, Z. Q.; Li, M.; Li, X.; Liu, X. M.; Ma, F.; Wu, S. L.; Yeung, K. W. K.; Han, Y.; Chu, P. K. Antibacterial Activity of Silver Doped Titanate Nanowires on Ti Implants. ACS Appl. Mater. Interfaces 2016, 8, 16584-16594. (23) Li, M.; Liu, X. M.; Xu, Z. Q.; Yeung, K. W. K.; Wu, S. L. Dopamine Modified Organic-Inorganic Hybrid Coating for Antimicrobial and Osteogenesis. ACS Appl. Mater. Interfaces 2016, 8, 33972−33981. (24) Ray, P. C.; Khan, S. A.; Singh, A. K.; Senapati, D.; Fan, Z. Nanomaterials for 41 ACS Paragon Plus Environment


Targeted Detection and Photothermal Killing of Bacteria. Chem. Soc. Rev. 2012, 41, 3193-3209. (25) Mohammadniaei, M.; Lee, T.; Bharate, B. G.; Yoon, J.; Choi, H. K.; Park, S. J.; Kim, J.; Kim, J.; Choi, J. W. Bifunctional Au@Bi2Se3 Core-Shell Nanoparticle for Synergetic Therapy by SERS-Traceable AntagomiR Delivery and Photothermal Treatment. Small 2018, 14, e1802934. (26) Zhang, X. B.; Sun, B. J.; Zuo, S. Y.; Chen, Q.; Gao, Y. L.; Zhao, H. Q.; Sun, M. C.; Chen, P. Y.; Yu, H.; Zhang, W. J.; Wang, K. Y.; Zhang, R. S.; Kan, Q. M.; Zhang, H. T.; He, Z. G.; Luo, C.; Sun, J. Self-Assembly of a Pure Photosensitizer as a Versatile Theragnostic Nanoplatform for Imaging-Guided Antitumor Photothermal Therapy. ACS Appl. Mater. Interfaces 2018, 10, 30155-30162. (27) Weissleder R. A Clearer Vision for in vivo Imaging. Nat. Biotech. 2001, 19, 316-317. (28) Yoo, D. W.; Jeong, H. Y.; Noh, S. H.; Lee, J. H.; Cheon, J. W. Magnetically Triggered Dual Functional Nanoparticles for Resistance-free Apoptotic Hyperthermia. Angew. Chem. Int. Ed. 2013, 52, 13047-13051. (29) Yang, Y.; Zhu, W. J.; Dong, Z. L.; Chao, Y.; Xu, L.; Chen, M. W.; Liu, Z. 1D Coordination Polymer Nanofibers for Low-Temperature Photothermal Therapy. Adv. Mater. 2017, 29, 1703588. (30) Zhang, Y. J.; Zhan, X. L.; Xiong, J.; Peng, S. S.; Huang, W.; Joshi, R.; Cai, Y.; Liu, Y. L.; Li, R.; Yuan, K.; Zhou, N. J.; Min, W. P. Temperature-dependent Cell Death Patterns Induced by Functionalized Gold Nanoparticle Photothermal Therapy 42 ACS Paragon Plus Environment

Page 42 of 49

Page 43 of 49 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


in Melanoma Cells. Sci. Rep. 2018, 8, 8720. (31) Prasad, P.; Gordijo, C. R.; Abbasi, A. Z.; Maeda, A.; Lp, A.; Rauth, A. M.; Dacosta, R. S.; Wu, X. Y. Multifunctional Albumin-MnO2 Nanoparticles Modulate Solid Tumor Microenvironment by Attenuating Hypoxia, Acidosis, Vascular Endothelial Growth Factor and Enhance Radiation Response. ACS Nano 2014, 8, 3202-3212. (32) Fan, W. P.; Bu, W. B.; Shen, B.; He, Q. J.; Cui, Z. W.; Liu, Y. Y.; Zheng, X. P.; Zhao, K. L.; Shi, J. L. Intelligent MnO2 Nanosheets Anchored with Upconversion Nanoprobes

for

Concurrent

pH-/H2O2-Responsive

UCL

Imaging

and

Oxygen-Elevated Synergetic Therapy. Adv. Mater. 2015, 27, 4155-4161. (33) Yuan, Y. X.; Wu, S. F.; Shu, F.; Liu, Z. H. An MnO2 Nanosheet as a Label-free Nanoplatform for Homogeneous Biosensing. Chem. Commun. 2014, 50, 1095-1097. (34) Wang, C. X.; Zhai, W. Y.; Wang, Y. X.; Yu, P.; Mao, L. Q. MnO2 Nanosheets Based Fluorescent Sensing Platform with Organic Dyes as a Probe with Excellent Analytical Properties. Analyst 2015, 140, 4021-4029. (35) Gordijo, C. R.; Abbasi, A. Z.; Amini, M. A.; Lip, H. Y.; Maeda, A.; Cai, P.; O'Brien, P. J.; DaCosta, R. S.; Rauth, A. M.; Wu, X. Y. Design of Hybrid MnO2-Polymer-Lipid Nanoparticles with Tunable Oxygen Generation Rates and Tumor Accumulation for Cancer Treatment. Adv. Funct. Mater. 2015, 25, 1858-1872. (36) Kim, S. C.; Ahn, S. M.; Lee, J. S.; Kim, T. S.; Min, D. H. Functional Manganese Dioxide Nanosheet for Targeted Photodynamic Therapy and Bioimaging in vitro and in vivo. 2D Mater. 2017, 4, 025069. 43 ACS Paragon Plus Environment


(37) Liu, Z.; Zhang, S. J.; Lin, H.; Zhao, M. L.; Yao, H. L.; Zhang, L. L.; Peng, W. J.; Chen, Y. Theranostic 2D Ultrathin MnO2 Nanosheets with Fast Responsibility to Endogenous Tumor Microenvironment and Exogenous NIR Irradiation. Biomaterials 2018, 155, 54-63. (38) Wang, Y. Z.; Song, Y. J.; Zhu, G. X.; Zhang, D. C.; Liu, X. W. Highly Biocompatible BSA-MnO2 Nanoparticles as an Efficient Near-infrared Photothermal Agent for Cancer Therapy. Chinese Chemical Letters 2017. (39) Xie, X. Z.; Mao, C. Y.; Liu, X. M.; Tan, L.; Cui, Z. D.; Yang, X. J.; Zhu, S. L.; Li, Z. Y.; Yuan, X. B.; Zheng, Y. F.; Yeung, K. W. K.; Wu, S. L. Tuning the Bandgap of Photo-Sensitive Polydopamine/Ag3PO4/Graphene Oxide Coating for Rapid, Noninvasive Disinfection of Implants. ACS Cent. Sci. 2018, 4, 724-738. (40) Roper, D. K.; Ahn, W.; Hoepfner, M. Microscale Heat Transfer Transduced by Surface Plasmon Resonant Gold Nanoparticles. J. Phys. Chem. C 2007, 111, 3636-3641. (41) Lu, N.; Huang, P.; Fan, W. P.; Wang, Z. T.; Liu, Y. J.; Wang, S.; Zhang, G. F.; Hu, J. K.; Liu, W. F.; Niu, G.; Leapman, R. D.; Lu, G. M.; Chen, X. Y. Tri-stimuli-responsive Biodegradable Theranostics for Mild Hyperthermia Enhanced Chemotherapy. Biomaterials 2017, 126, 39-48. (42) Feng, Z. Z.; Liu, X. M.; Tan, L.; Cui, Z. D.; Yang, X. J.; Li, Z. Y.; Zheng, Y. F.; Yeung, K. W. K.; Wu, S. L. Electrophoretic Deposited Stable Chitosan@MoS2 Coating with Rapid in Situ Bacteria-Killing Ability Under Dual-Light Irradiation. Small 2018, 14, 1704347. 44 ACS Paragon Plus Environment

Page 44 of 49

Page 45 of 49 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


(43) Li, W. R.; Xie, X. B.; Shi, Q. S.; Zeng, H. Y.; Ou-Yang, Y. S.; Chen, Y. B. Antibacterial Activity and Mechanism of Silver Nanoparticles on Escherichia coli. Appl. Microbiol. Biotechnol. 2010, 85, 1115−1122. (44) Zhao, R. T.; Lv, M.; Li, Y.; Sun, M. X.; Kong, W.; Wang, L. H.; Song, S. P.; Fan, C. H.; Jia, L. L.; Qiu, S. F.; Sun, Y. S.; Song, H. B.; Hao, R. Z. Stable Nanocomposite Based on PEGylated and Silver Nanoparticles Loaded Graphene Oxide for Long-Term Antibacterial Activity. ACS Appl. Mater. interfaces 2017, 9, 15328-15341. (45) Li, Y. L.; Chen, L.; Liu, Y. Y.; Zhang, Y.; Liang, Y. X.; Mei, Y. X. Anti-inflammatory Effects in a Mouse Osteoarthritis Model of a Mixture of Glucosamine and Chitoligosaccharides Produced by Bi-enzyme Single-step Hydrolysis. Sci. Rep. 2018, 8, 5624. (46) Wang, Y. X.; Sun, H. Q.; Ang, H. M.; Tade, M. O.; Wang, S. B. Facile synthesis of hierarchically structured magnetic MnO2/ZnFe2O4 hybrid materials and their performance in heterogeneous activation of peroxymonosulfate. ACS Appl. Mater. interfaces 2014, 6, 19914-19923. (47) Baranwal, A.; Chiranjivi, A. K.; Kumar, A.; Dubey, V. K.; Chandra, P. Design of

Commercially

Comparable

Nanotherapeutic

Agent

Against

Human

Disease-causing Parasite, Leishmania. Sci. Rep. 2018, 8, 8814. (48) Jin, S. H.; Kim, S. M.; Lee, S. Y.; Kim, J. W. Synthesis and Characterization of Silver Nanoparticles Using a Solution Plasma Process. J. Nanosci. Nanotechnol. 2014, 14, 8094-8097. 45 ACS Paragon Plus Environment


(49) Xiao, W.; Wang, D. L.; Lou, X. W. Shape-controlled Synthesis of MnO2 Nanostructures with Enhanced Electrocatalytic Activity for Oxygen Reduction. J. Phys. Chem. C 2009, 114, 1694-1700. (50) Shameli, K.; Ahmad, M. B.; Jazayeri, S. D.; Sedaghat, S.; Shabanzadeh, P.; Jahangirian, H.; Mahdavi, M.; Abdollahi, Y. Synthesis and Characterization of Polyethylene Glycol Mediated Silver Nanoparticles by the Green Method. Int. J. Mol. Sci. 2012, 13, 6639-6650. (51) Mahamallik, P.; Saha, S.; Pal, A. Tetracycline Degradation in Aquatic Environment by Highly Porous MnO2 Nanosheet Assembly. Chem. Eng. J. 2015, 276, 155-165. (52) Jin, J. C.; Xu, Z. Q.; Dong, P.; Lai, L.; Lan, J. Y.; Jiang, F. L.; Liu, Y. One-Step Synthesis of Silver Nanoparticles Using Carbon Dots as Reducing and Stabilizing Agents and Their Antibacterial Mechanisms. Carbon 2015, 94, 129−141. (53) Cui, J. B.; Li, Y. J.; Liu, L.; Chen, L.; Xu, J.; Ma, J. W.; Fang, G.; Zhu, E. B.; Wu, H.; Zhao, L. X.; Wang, L. Y.; Huang, Y. Near-Infrared Plasmonic-Enhanced Solar Energy Harvest for Highly Efficient Photocatalytic Reactions. Nano letters 2015, 15, 6295-6301. (54) Zhu, G. X.; Bao, C. L.; Liu, Y. J.; Shen, X. P.; Xi, C. Y.; Xu, Z.; Ji, Z. Y. Self-regulated route to ternary hybrid nanocrystals of Ag-Ag2S-CdS with near-infrared photoluminescence and enhanced photothermal conversion. Nanoscale 2014, 6, 11147-11156. (55) Tan, H. L.; Ma, R.; Lin, C. C.; Liu, Z. W.; Tang, T. T. Quaternized Chitosan as 46 ACS Paragon Plus Environment

Page 46 of 49

Page 47 of 49 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


an Antimicrobial Agent: Antimicrobial Activity, Mechanism of Action and Aiomedical Applications in Orthopedics. Int. J. Mol. Sci. 2013, 14, 1854-1869. (56) Li, P.; Poon, Y. F.; Li, W. F.; Zhu, H. Y.; Yeap, S. H.; Cao, Y.; Qi, X. B.; Zhou, C. C.; Lamrani, M.; Beuerman, R. W.; Kang, E. T.; Mu, Y. G.; Li, C. M.; Chang, M. W.; Leong, S. S.; Chan-Park, M. B. A Polycationic Antimicrobial and Biocompatible Hydrogel with Microbe Membrane Suctioning Ability. Nat. Mater. 2011, 10, 149-156. (57) Tan, L.; Li, J.; Liu, X. M.; Cui, Z. D.; Yang, X. J.; Zhu, S. L.; Li, Z. Y.; Yuan, X. B.; Zheng, Y. F.; Yeung, K. W. K.; Pan, H. B.; Wang, X. B.; Wu, S. L. Rapid Biofilm Eradication on Bone Implants Using Red Phosphorus and Near-Infrared Light. Adv. Mater. 2018, 30, e1801808. (58) Ding, X.; Yang, C.; Lim, T. P.; Hsu, L. Y.; Engler, A. C.; Hedrick, J. L.; Yang, Y. Y. Antibacterial and Antifouling Catheter Coatings Using Surface Grafted PEG-b-cationic Polycarbonate Diblock Copolymers. Biomaterials 2012, 33, 6593-6603. (59) Su, H. L.; Chou, C. C.; Hung, D. J.; Lin, S. H.; Pao, I. C.; Lin, J. H.; Huang, F. L.; Dong, R. X.; Lin, J. J. The Disruption of Bacterial Membrane Integrity Through ROS Generation Induced by Nanohybrids of Silver and Clay. Biomaterials 2009, 30, 5979-5987. (60) Deng, R. R.; Xie, X. J.; Vendrell, M.; Chang, Y. T.; Liu, X. G. Intracellular Glutathione Detection Using MnO2-nanosheet-modified Upconversion Nanoparticles. J. Am. Chem. Soc. 2011, 133, 20168-20171. 47 ACS Paragon Plus Environment


(61) Lin, L. S.; Song, J. B.; Song, L.; Ke, K. M.; Liu, Y. J; Zhou, Z. J.; Shen, Z. Y.; Li, J.; Yang, Z.; Tang, W.; Niu, G.; Yang, H. H.; Chen, X. Y. Simultaneous Fenton-like Ion Delivery and Glutathione Depletion by MnO2 -Based Nanoagent to Enhance Chemodynamic Therapy. Angew. Chem. Int. Ed. 2018, 57, 4902-4906. (62) Cui, Y.; Zhao, Y. Y.; Tian, Y.; Zhang, W.; Lu, X. Y.; Jiang, X. Y. The Molecular Mechanism of Action of Bactericidal Gold Nanoparticles on Escherichia coli. Biomaterials 2012, 33, 2327-2333. (63) Li, Y.; Liu, X. M.; Tan, L.; Cui, Z. D.; Yang, X. J.; Zheng, Y. F.; Yeung, K. W. K.; Chu, P. K.; Wu, S. L. Rapid Sterilization and Accelerated Wound Healing Using Zn2+ and Graphene Oxide Modified g-C3N4 under Dual Light Irradiation. Adv. Funct. Mater. 2018, 28, 1800299. (64) Xiu, Z. M.; Zhang, Q. B.; Puppala, H. L.; Colvin, V. L.; Alvarez, P. J. J. Negligible Particle-Specific Antibacterial Activity of Silver Nanoparticles. Nano Lett. 2012, 12, 4271−4275. (65) Yuan, W. H.; Gu, Y. J.; Li, L. Green Synthesis of Graphene/Ag Nanocomposites. Appl. Surf. Sci. 2012, 261, 753−758.


Page 48 of 49

Page 49 of 49 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


TOC Figure


CS

Recommend Documents