Graphene oxide synergistically enhances antibiotic efficacy in

Subscriber access provided by Iowa State University | Library

Article

Graphene oxide synergistically enhances antibiotic efficacy in Vancomycin resistance Staphylococcus aureus Vimal Singh, Vinod Kumar, Sunayana Kashyap, Ajay Vikram Singh, vimal kishore, Metin Sitti, Preeti S Saxena, and Anchal Srivastava ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00757 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 4, 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 31 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 Bio Materials

162x87mm (150 x 150 DPI)

ACS Paragon Plus Environment

ACS Applied Bio Materials 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 2 of 31

Graphene oxide synergistically enhances antibiotic efficacy in Vancomycin resistance Staphylococcus aureus Vimal Singh1, Vinod Kumar1, Sunayana Kashyap1, Ajay Vikram Singh2, Vimal Kishore2, Metin Sitti2, Preeti S Saxena1*, Anchal Srivastava3* 1Department

2Physical

of Zoology, Banaras Hindu University, Varanasi-22005, India

Intelligence Department, Max Planck Institute for Intelligent Systems, 70569 Stuttgart,

Germany 3Department

of Physics, Banaras Hindu University, Varanasi-22005, India

Corresponding author: [email protected], [email protected] ABSTRACT Current study highlights a new polyvalent inhibitor approach based on Vancomycin-conjugated with Graphene Oxide (Van@GO) against Vancomycin resistant Staphylococcus aureus (VRSA) strain. Physicochemical characteristics of the prepared Van@GO composites were studied using UV-Vis and FTIR spectroscopy techniques. Characterization results confirm the attachment of Vancomycin to the Graphene Oxide. A significant inhibition of VRSA growth is achieved by Vancomycin when presented as Van@GO. The polyvalent inhibition activity of Van@GO was characterized by performing bacteriological experiments along with scanning electron microscopy. Results clearly exhibit the enhanced inhibition activity of Van@GO than Vancomycin alone against VRSA. High surface area of GO facilitates high loading and multivalent interaction of conjugated Vancomycin leading to polyvalent inhibition. Further, we found that Van@GO significantly reduces the motility of VRSA via inducing oxidative stress


1

Page 3 of 31 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


compare with untreated samples. Our findings highlight the importance of Van@GO as an effective polyvalent inhibition recipe for VRSA.. Keywords: polyvalent inhibitors, graphene oxide, vancomycin, multivalent interactions 1. Introduction In the last two decades, the increasing load of communicable diseases with heavy use of antibiotics, have resulted into the progress and persistence of drug-resistant infections.1 The continuous emergence of drug resistance bacteria against the most potent antibiotics are presenting a serious threat to the public health. Also, it is resulting to increased chances of serious illness leading to death in most of the cases, with an extra expenditure of >20 billion USD annually for developing an efficient health care system.1,2 A large number of infectious bacterial strains has developed antibiotics opposition, for example, Pseudomonas aeruginosa, methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococci (VREs) along with those infecting to the immunocompromised conditions for eg. HIV/AIDS.3,4. Among others, more than 90% of Staphylococcus strains have acquired the resistance to several potent antibiotics drugs like penicillin, methicillin, etc. which is causing serious concern.5,6 Moreover, the huge and persistent decline in the supply of endorsed antibiotics in the previous decade has added to the undeniably compromising circumstance.7 Hence, there is an earnest need to search for an efficient therapeutic means to overcome the existing challenges of bacterial resistance. So as to beat the developing pattern of resistance, a strategy is required so as to maximize the therapeutic value of antibiotics which can serve the purpose up to certain extent. In the present situation, nanomaterials and nanoparticels have emerged as a versatile tool to enhance the medicinal viability or to battle against Multi Drug Resistance bacterial strains, commonly known as MDR strains.3,8–14 The application of nanoparticles (NPs) in battle against


2


Page 4 of 31

MDR may be due to single or multiple properties of NPs: (i) The size range of the NPs commensurate with biomolecular and bacterial cellular systems and hence, can effectively pass through cell membrane as a carrier moiety, (ii) NPs, have the enhanced surface area enabling presentation of multivalenct antibiotic on surface to enhance interactions with target bacteria, (iii) NPs can explicitly assault biological targets after alteration with target particles.15 The concept of nanoparticles as an antimicrobial agent is not new. Till date, a number of nanoparticles and their composites have been explored as a potent antimicrobial agent.16–21 Among the various nanoparticles, silver nanoparticles have gained considerable attention as an effective antibacterial agent.16,22–24 Furthermore, silver nanoparticles in mix with several antibiotics have proven more suitable bactericidal impact against drug resistant pathogenic bacteria.25 Complexes of NPs and drug molecules could serve as an alien concept which bacteria could not recognize as a threat and thus bypass the problem of resistance. One of the possible explanations of the enhanced antibacterial activities as shown in above cases could be assigned to the “NPs facilitated polyvalent inhibition effect of drug molecules”. In recent years, Graphene along with its chemical derivatives viz. Graphene oxide (GO) and others have been largely explored for various applications in basic sciences.26–31 Some studies suggest that GO, its derivatives, and their composites with other nanoparticles can possess antibacterial as well as antifungal activities.32,33 Vancomycin, a unique glycopeptide antibiotic, is unrelated to any currently available antibiotics. It is recognized as a symbolic antibiotic. It has been a preferred antibiotic for the treatment of infections due to deadly streptococcal and staphylococcal strains that have developed resistance to MRSA.34 Vancomycin acts towards gram-positive microorganisms just, by restraining the means in murein (peptidoglycan) bio-synthesis and in assembly of NAM-NAG-polypeptide into


3

Page 5 of 31 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 developing peptidoglycan chain. It has been found that Vancomycin interact partially with the bacteria due to lack of sufficient reacting sites for bacterial attachment.35 In other words, effectiveness of Vancomycin against the bacterial system is very much limited due to its meager affinity to the reprogrammed cell wall structure. These limitations of Vancomycin can be largely overcome by using nanomaterials (for e.g. GO) as suitable career. GO as a carrier could significantly enhance the therapeutic efficacy of Vancomycin: (i) Owing to the large surface area, GO will provide high local concentration of Vancomycin, (ii) facilitate multivalent interaction of Vancomycin to target bacteria. Thus, GO offers a unique way to increase the therapeutic potential of Vancomycin by enabling a strong interaction of Vancomycin with cell walls of VRSA resulting in significant bactericidal activity. Considering the above facts, present study is aimed to develop an exclusive polyvalent inhibition approach relying on the unique features of GO employed for not only high loading of molecules of vancomycin but also for presentation of the drug molecules to VRSA, in such a way to maximize their inhibitory effect, thus moving a step forward in the direction of developing effective therapeutic measures for VRSA. 2. Experimental Section 2.1. Chemicals/reagents Kish graphite flakes had been procured from NGS Naturgraphit GmbH (Germany) and used as received. Orthophosphoric acid (H3PO4), Hydrochloric acid (HCl), Sulphuric acid (H2SO4) and Ethanol were procured from Merck Limited, Mumbai. Sodium chloride (NaCl), glutaraldehyde, potassium permanganate (KMnO4), sodium dihydrogen phosphate, dimethyl sulfoxide (DMSO) and propidium iodide (PI) were purchased from Qualigens (lndia). Vancomycin hydrochloride hydrate, Buffers (Tris and PBS), and Concanavaline A-FITC conjugate (Con-A-FITC) was


4


Page 6 of 31

purchased from Sigma Aldrich Chemical Private Limited, Mumbai, India. . SOD Kit was purchased from Enzolife sciences, Germany. Assay KIT for the detection and estimation of Reactive Oxygen molecules was procured from Abcam, USA. Luria broth and agar medium was purchased from Himedia Pvt. Ltd. The bacterial strain of Vancomycin resistant Staphylococcus aureus was generously gifted by Prof. GopalNath, Department of Microbiology, Institute of Medicine Science, Banaras Hindu University, Varanasi, India. All the reagents used for this experiment were of analytical grade. 2.2. Synthesis of Graphene Oxide GO was synthesized following the method reported by Marcano et al.36 In brief, Graphite flakes were allowed to react with concentrated sulphuric acid and ortho-phosphoric acid (V:V::9:1) in an ice cooled flask and stirred. Potassium permanganate was then added over an hour period with care, maintaining the temperature at ~40 °C. The resulting solution was stirred at50 °C for 12 h. After stirring, the solution was allowed to cool down to room temperature, thereafter, ice was addedwith H2O2 (30%). Finally, the mixture was filtered and the residue was washed with distilled water, 30% HCl, and ethanol simultaneously in three successive steps. The GO material was coagulated by ether and filtered. The solid obtained on the filter was dried overnight in oven. 2.3. Conjugation of Vancomycin to Graphene Oxide (Van@GO) Vancomycin conjugated Graphene Oxide (referred as Van@GO in this manuscript) was obtained following the protocol reported earlier with slight modification.37,38 Before the covalent binding of vancomycin to the –COOH functional groups of GO, an activation of its –COOH groups was done by using a coupling agent, EDC (10 µL 1 mg mL-1) and an activator i.e. NHS (10 µL 1 mg mL-1). A 1 mg mL-1 stock solution of GO was prepared by ultrasonication at RT. A 10 mg of vancomycin was dissolved in 1 mL of sterile distilled water to obtain a working vancomycin


5

Page 7 of 31 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


solution of 10 mg/mL-1. The -COOH present in GO were activated with an equimolar solution of EDC and NHS for 30 min. Activated GO solution (1 mg mL−1) was mixed with vancomycin solution in a volume to volume ratio of 2:1 at room temperature overnight to complete the reaction. Finally, a stable suspension of Vancomycin loaded GO (Van@GO) was obtained and stored at 4 °C until use. Vancomycin binds covalently to the GO, through its free –NH2 groups to the free –COOH groups of GO. Scheme 1 shows the conjugation chemistry of vancomycin to GO. 2.4. Instrumentation and measurements For characterization of GO, various characterization tools like UV-Vis, Fourier Transform Infrared (FT-IR), Raman and XRD were used. To confirm the covalent conjugation of Vancomycin onto GO with an amide bondFTIR (Perkin Elmer) was used. The optical absorbance of GO, Vancomycin and Van@GO were acquired by using a UV-vis-NIR spectrometer

(Perkin-Elmer).

The

electron

microscopy

images

were

taken

using

Zeiss EVO® LS 10 scanning electron microscope (SEM), operated at 3.89 × 10 – 5 Torr, 5 kV in inert atmosphere. 2.5. Antibacterial tests 2.5.1. Culture and maintenance of bacterial growth Vancomycin resistant Staphylococcus aureus (VRSA) strains were used for testing the antibacterial properties of Van@GO. A culture of VRSA were grown and maintained on LB media and agar plates. The bacterial strain was grown overnight at 37 oC using agar media. The inoculum for antibacterial measures was prepared from actively growing bacteria (Logarithmic stage). The bacteria concentration was controlled by estimating optical density at 600 nm


6


Page 8 of 31

(OD600). The bacterial culture media being yellow in colour exhibits high absorbance ~ 400-500 nm but very low absorbance ~600 nm. This makes quantification of bacterial cell density accurate at OD600. 2.5.2. Bacterial growth kinetics Antibacterial effect of Van@GO was studied by measuring the time dependent absorbance of treated bacteria growth at 600 nm. For growth kinetic study, VRSA strains were grown in LB media and incubated at 37 oC on an orbital shaker at a rotation speed of 200 rpm for ~6 h. A diluted bacterial concentration corresponding to OD 0.1 (Abs600) was used for rest of the study. A 20 µL of the bacterial suspension (initial concentration of 105-106 colony forming unit (CFU) per mL) was added to each well of a microtiter plate along with PBS (20 µL) as control. A 20 µL of each Vancomycin, GO, and Van@GO were added to respective wells and allowed to grow at 37 oC on an orbital shaker (rotation speed of 200 rpm). Concentration dependent bactericidal effect of Van@GO was investigated by exposing it to bacterial culture at 10, 12.5, 15 and 20 mg mL-1 of concentrations. The bacterial viability was monitored at regular intervals by taking absorbance at 600 nm. All studies were done in triplicates. 2.5.3. Evaluation of bactericidal property by Kirby-Bauer disk diffusion method This approach20 was opted to evaluate the antimicrobial activity of Van@GO against VRSA. In this technique, sterilized filter paper circular discs (6.0 mm in diameter) soaked with different concentrations of GO, Vancomycin and Van@GO (0–200µg/disc) were used. Overnight grown 100 µL cultures of VRSA were spread-plated on Luria broth agar plates by swabbing method. The impregnated discs were then exposed to the bacterial culture grown on the agar surface. All culture plates with bacteria and treated samples were overnight incubated at 37 oC. Bactericidal efficacies of different systems were characterized by measuring the zone of inhibition and by


7

Page 9 of 31 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


SEM. Mean values were utilized for the figuring of the inhibition zone, which was the proportion of antibacterial activity of the studied samples. 2.6. SEM analysis of treated S. aureus strains The effect of GO, Vancomycin and Van@GO on VRSA were examined by SEM. The SEM samples were prepared by drop-casting of 2 µL fixed (overnight with 4% glutaraldehyde) bacterial ting of suspensions, with and without GO, Vancomycin and Van@GO on silicon wafer respectively. The samples were coated with gold followed by analysis under SEM. 2.7. Biofilm formation and cell motility tracking via nucleic acid labeling with fluorescent marker The protocol for the fluorescent staining to bacterial cells was followed from our previous work.39 In brief, a subculture of bacteria was from stock preserved at -20 oC was established for 12 hours. A total of 100 µL of this culture was adjusted to an optical density of 0.2@Abs600 nm and added to the 24 well cell culture plate with circular glass cover slips (CGS) place at the bottom of the well coated with Van, GO, and Van@GO. After day oneincubation at room temperature (RT), the substrate of CGS with biofilm was rinsed with 125 mM saline in order to eliminate any earlier non-adherent bacteria. A final volume of 100 µL Tris-buffered saline (TBS) was freshly prepared and further added to the bacterial culture. All the substrates (GO Van and Van@GO) were incubated at RT for 24 hrs. After the biofilms growth, the substrates were rinsed with 125 mM NaCl and 5 µM of Con-A-FITC (excitation:488 nm, emissions: 500–600 nm range) was added to TBS containing cell permeable red fluorescent propidium iodide with 1∶500 dilution in 5 mM in DMSO (Invitrogen, CA). The bacterial culture plate containing biofilm samples was incubated at RT for half an hour in the dark in order to enable the dye diffusion into the attached biofilm. Samples imaging and biofilm structural properties (biovolume, 3D


8


Page 10 of 31

morphology) were determined in-situ via Nikon Eclipse Ti Confocal Microscope confocal laser scanning microscope with Cool SNAP HQ2 CCD camera (Photometrics, AZ). To minimize the photobleaching for fluorescent dye, the CCD camera was connected to the Yokogawa CSU-W1 spinning disk at 40x or 100 x magnification of oil/water immersion objective (zoom 1:5, 0.8 N.A.). The average z-stacks slices of 0.5 µm were captured in horizontal plane using z-section mode from each. A maximum of 10 stacks were collected at different field of view for each sample. Using the IMARIS 7.0, Bitplane's core software (Saint Paul, Minnesota, USA), we performed three-dimensional (3D) reconstruction of projections of biofilms structure with Easy 3D function. For bacterial cell tracking, fluorescent images were collected every 5 minutes for 12 hours 2.8. SOD and ROS assay SOD is considered as an important oxidative stress marker, it plays a vital role in scavenging of superoxide radicals into H2O2. Thus, the SOD activity of VRSA treated with Van@GO was estimated by using a SOD estimation Kit (ELS-SOD, Enzolife sciences, Germany). Treated cultures were centrifuged at 5,000 rpm for 5 min at 24 °C and washed using TBS. Bacterial cells were re-suspended with the same volume of TBS buffer and OD adjusted to the OD600 value in the treated group. The culture solution was centrifuged at 5000 rpm for 5 min, and the supernatant was collected. The cell suspensions to remove the cellular debris were sonicated in ice bath for 3 times (0 s off and 15 seconds on). We followed the instructions from manufacturer manual supplied with SOD kit. The reactive oxygen species (ROS) activity was measured with cellularROS Detection Assay KIT based on cell permeant component 2’,7’ –dichlorofluorescin diacetate (DCFDA) fluorescence quantification (Abcams Cat # 113851). In short, overnight culture of bacterial


9

Page 11 of 31 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 were stained with fluorescent ROS detection dye at 37 0C for 45 minutes and washed with 1X PBS. Subsequently, these cells were treated with either GO or Van@GO with a control culture without any treatment was established for 1 hour period. Fluorescence measurement (Ex/Em=485/535 nm) in a standard microplate reader (SYNERGY, Biotek, USA) after background corrections, provides ROS change as percentage of control. 2.9. Statistical analysis The analyses were performed in triplicates. The outcomes were expressed as the mean of the values acquired from three autonomous investigations. Contrasts in mean, among control and bacteria treated with each test nanoparticles were examined utilizing Student's t test via GraphPad Prism 5 program and ( p < 0.05 ) was considered statistically significant. 3. Results and discussion Vancomycin is a glycopeptide that has an exclusive approach of action different from that of penicillin and cephalosporin. It hinders the second phase of cell wall synthesis in susceptible bacteria. Increasing opposition of S. aureus to vancomycin in developed and developing nations is disturbing. The first report of VRSA appeared in 1996.40 Later in 2002, in USA, fully vancomycin resistant strain of S. aureus was reported.41 Within a few years of onset of the antibiotics era, resistance development in S. aureus against vancomycin has prompted renewed efforts for new and innovative anti-VRSA drugs. In the present study, we have utilized a carbon based nanomaterial, graphene oxide, which binds with Vancomycin as prototype of polyvalent inhibitors against VRSA. 3.1.

Characterizations of GO


10


Page 12 of 31

To affirm that the synthesized GO utilized in this investigation had comparable properties as recently revealed, UV−vis spectroscopy, XRD and SEM was utilized for characterization. Further, the synthesized GO was also characterized by performing Raman spectroscopy, which is a non-destructive technique and much relevant for carbon based materials. The Raman spectrum of GO (Figure 1a) exhibits well documented peak of D band at 1350 cm-1 which arises due the sp3 defects. Another peak of G band at 1597 cm-1 in Raman spectrum of GO is attributed to the in plane mode of vibrations of sp2 bonded carbon atoms and a doubly degenerated phonon mode (E2g symmetry) at the centre of Brillouin zone.42 Calculated intensity ratio of the D and G band (Id/Ig ratio) was found to be 1.03. Previous examinations have shown that Id/Ig ratio increases with the extent of disorder in graphitic nanomaterials, and vanishes in completely imperfection free graphite,43,44 which is a proportion of the disorder as articulated by the sp2/sp3 carbon ratio.45 The strong D band in the present GO indicates that the degree of graphitization is high. Further, to probe the exfoliation of GO, its X-ray diﬀraction (XRD) was documented in the 2θ range value of 5⁰–60⁰ and the result has been shown in Figure 1b. The result reveals an intense peak at 2θ =11.4⁰ (002) corresponding to an interlayer spacing of ~0.75 nm, indicating the presence of oxygen containing groups, which facilitates the easy hydration and exfoliation of GO in aqueous media. Another peak found at 42.17⁰ (100) corresponds to reflection plane of GO. The SEM pictures (Figure 1c) uncovered that the GO material comprises of arbitrarily collected, thin, folded sheets intently connected with one another, framing a cluttered strong solid. Presence of negative charge on the surface of GO and positive charge on vancomycin was affirmed by zeta potential estimation as appeared in Figure 1d. Also a SEM image of Van@GO has been presented in Figure S1.


11

Page 13 of 31 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) Raman spectra presenting shifts in the region of the D, G and 2D peaks, (b) XRD patterns, (c) SEM image of GO and (d) Zeta potential.

3.2.

Conjugation of Vancomycin with Graphene oxide (Van@GO)

For the synthesis of Van@GO, carboxylic functional group of GO was taken advantage of. Vancomycin was conjugated onto GO via amide linkages using coupling chemistry between carboxylic groups (–COOH) of GO and amine groups (–NH2) of Vancomycin using crosslinking

agents

i.e.

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

(EDC)

and

N-

hydroxysuccinamide (NHS). 3.3.

Characterization of Van@GO


12


Page 14 of 31

The mechanism for the attachment of Vancomycin on GO surface has been illustrated in Scheme 1. EDC activated –COOH functional groups of GO form an amide bond with available free -NH2 groups of Vancomycin. The formation of amide bond provides stability to the Van@GO.

Scheme 1. Schematic representation for the synthesis of Van@GO where (a) is graphene oxide, (b) is vancomycin and (c) is Van@GO.


13

Page 15 of 31 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


Adequate GO guarantees that all Vancomycin molecules are totally bound to the surface of GO on complete reaction. Aqueous dispersion of GO (1 mg mL-1) was studied using UV−vis absorption spectrum, as presented in Figure 2a. It showed a maximum absorption peak at 225 nm due to the π−π* transition of aromatic C=C bonds and a shoulder around 300 nm due to the n−π* change of C-O bonds, which is reliable with past reports.46,47 Vancomycin possess characteristic absorption peak at 280 nm (Figure 2a). Further, loading of GO with Vancomycin leads to shift in characteristics absorption peak of GO from 225 nm to a new peak position at 279 nm, close to absorption peak of vancomycin (Figure 2a).48–50 This suggests that the interactions of free carboxyl groups present on the surface of GO and the amine groups in Vancomycin facilitated the conjugation reactions.

Figure 2. (a) UV-Visible spectra of GO, Vancomycin and Van@GO; (b) FTIR spectra of GO, Vancomycin and Van@GO Further, FT-IR study was performed to probe the interaction between GO and vancomycin (Figure 2b). The FT-IR spectrum of GO reveals characteristic vibrational peaks corresponding to O–H stretching at 775 cm-1, 1063 cm-1 and 3425 cm-1, C=O bands at 1393 cm-1 and 1633 cm-1 and C-O-C bands 1224 cm-1 and 1315 cm-1 which were similar to the previous


14


Page 16 of 31

reports.51,52 Vancomycin shows a distinguishable characteristic peak of NH2 (1°-amine) at 1639 cm-1. Other representative peaks of Vancomycin observed are O-H stretching corresponding to 1397 cm-1 and 3187 cm-1, C-N bonds corresponding to 1030 cm-1, 1062 cm-1, 1124 cm-1 and O-C and C=O bonds corresponding to 1312 cm-1 and 1231 cm-1 respectively. However, the conjugation of Vancomycin onto GO surface leads to appearance of a new characteristic N-H (2°-amide) II band at 1543 cm-1, which approves the formation of Van@GO. Moreover, peaks conforming to C-N bond at 1097 cm-1, C-O-H bonding at 1416 cm-1, C-O (amide I) band at 1648 cm-1, CH2 at 2884 cm-1 and O-H (H-bonded) at 3404 cm-1 further confirm the attachment of Vancomycin on the surface of GO. 3.4.

Antibacterial Activity

Antibacterial activity of Van@GO against VRSA was estimated via the broth dilution approach as well as by disk diffusion assay. The growth profile of VRSA was utilized to sift post treatment bacterial viability. The growth kinetics of VRSA on post treatment with GO, Vancomycin, and Van@GO is presented in Figure 3a. The result clearly depicts that Van@GO has significant inhibiting effect on the growth of VRSA as compared to GO and Vancomycin alone. The enhanced propensity of Van@GO against VRSA is further supported by the obtained bacterial cell viability data (Figure 3b), calculated from the colony forming unit (CFU). The CFU results were as per growth kinetic study obtained for VRSA. The reduced cell viability after treatment with Van@GO verifies its enhanced antibacterial potential. Conjugating different concentration of Vancomycin on GO further confirmed that higher drug loading completely abolishes the bacterial density due enhanced synergistic effect of GO (supporting information Figure S2).


15

Page 17 of 31 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 3. Cytotoxicity assay of Van@GO for S. aureus using (a) optical density (OD600); (b) colony forming units (CFU) expressed as percentage of viable cells; (c) culture plate showing zone of inhibition and (d) Mean value of diameter of the zone of inhibition. Further, standard zone of inhibition assessment was also carried out to compare the anti VRSA activity of Van@GO, Vancomycin and GO. Optical images were shown in Figure 3c. Followed by treatment, the zones of inhibition were measured and are shown in Figure 3d. Van@GO has shown incredible antibacterial activity, hence a bigger zone of inhibition than that of Vancomycin alone. However, no antibacterial activity has been found in case of GO which showed similar zone of inhibition as shown by control. Vancomycin showed an inhibition zone


16


Page 18 of 31

with the diameter of 9 mm while Van@GO showed inhibition zone of 20 mm diameter. The results clearly reveal that Van@GO has huge potential to inhibit the growth of VRSA. 3.5.

Antibacterial activity characterized by SEM To further characterize the enhanced propensity of Van@GO, SEM imaging of the

samples treated with GO, Vancomycin and Van@GO were carried out. As shown in Figure 4a, untreated (control) VRSA cell are spherical in shape and healthy with intact cell wall. There was no evidence of membrane damage and collapse. Treatment of these cells with GO (Figure 4b) did not alter the morphologies of cells and thus, it can be concluded that GO is quite neutral towards the aforementioned bacterium. When VRSA cells were exposed to pristine Vancomycin (Figure 4c), it resulted into killing of VRSA but not as significant as caused by Van@GO (Figure 4d). The VRSA cells exposed to Van@GO exhibited a wide range of abnormalities.

Figure 4. SEM micrograph of the (a) controlled VRSA (b) interaction of VRSA with GO surface (c) interaction of VRSA with vancomycin (d) interaction of VRSA with Van@GO


17

Page 19 of 31 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 cells indicated broad membrane damage and most likely, spill out of the intracellular part causing shrinkage of cell finally cell lysis. The damaged morphology, together with cell breach, can be ascribed to the polyvalent inhibition activity of Van@GO. The plethora of Vancomycin molecules that were presented with the aid of GO to bacterial cell inhibited the cell wall formation leading to increased turgor pressure. Further, it is presumed that Van@GO facilitates high number of positively charged Vancomycin molecules for interaction with negatively charged bacteria that might have resulted in increased reactive oxygen species production.19,53 As a result, reactive oxygen species caused damage to the membrane integrity leading to cell death.19 Thus, it is believed that the polyvalent inhibition activity of Vancomycin in Van@GO bears immense potential to cope up with outbreak of resistant bacterial strains. Van@GO causes biofilm inhibition, oxidative stress and reduced motility of VRSA The investigation of bacterial colonization and multiplication on in-vivo embed surfaces is as basic as the investigation of eukaryotic cell collaborations for assessing biomaterials execution for biomedical applications. Despite its significance, to the top of our information, there are no studies devoted to understanding how Vancomycin supplemented with graphene oxide formulation could affect VRSA interactions and biofilm formation in vitro. Therefore, we studied VRSA biofilm characteristics on interaction with Van@GO. As shown in figure 5a-d, the different control groups show the differential inhibition of bacterial colonization as qualitatively determined by comparing live/dead cells in 3D reconstructed topography of biofilm on Van@GO. Untreated and Vancomycin treated bacterial cultures show similar propensity to colony growth except more live cells are found on in untreated samples as expected. Contrary to GO, Van@GO show high population of dead cells which could be attributed to enhanced propensity of vancomycin due to GO immobilization. Green 3D carpet like growth of VRSA on


18


Page 20 of 31

control culture is observed due to S. aureus exhibiting biofilm associated more exopolysaccharides (EPS) production compare to other control samples.54 In control culture, S. aureus secreted EPS binding with fluorescein isothiocyanate conjugated concanavalin A (ConAFITC) staining yields the green carpet like qualitative observation as shown in three dimensional reconstruction of biofilm shown in figure 5a-d. In control cultures, Imeris based 3D reconstruction conspicuously shows VRSA colonies encapsulated in EPS forming well dispersed profuse biofilm with fewer dead cells (figure. 5a). The VRSA grown directly on GO show scattered grass like FITC-positive stained EPS with intermittent zones without any colony (figure. 5b), however these patches are more prominent in Vancomycin treated cultures showing complete inhibition of growth (figure. 5c). The most prominent synergetic effect with scattered population of PI-positive dead cells can be seen in Van@GO samples (figure.5d). The inhibitory order follow: Van@GO > Vancomycin > GO > Untreated positive control. Figure 5e show mean speed calculated from VRSA treated with Van@GO versus different controls. From the graph, it is evident that cells treated with Van@GO exhibit a significant 3 fold decrease in motility in comparison with untreated controls cells and this variance was statistically significant (Student t-test, **p-value = 0.001 for control vs Van@GO and *p-value = 0.03 for control vs GO). The tracks extracted from time lapse movies recorded from control (MOVIE 1, SI) and Van@GO treated (MOVIE 2, SI) bacterial cultures support this notions qualitatively as very few motile cell tracks can be seen in Van@GO sample (figure 5f-g). Carbon or carbon family related NPs treatment to biological cultures are reported to cause oxidative stress, which predominately compromises the cellular motility.55


19

Page 21 of 31 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 5. (a-d) Image of 3D topographic reconstruction showing bacterial microcolonies encapsulated in EPS forming profuse biofilm on untreated, Vancomycin treated, GO and Van@GO substrate and scattered patches of microcolonies. (e-g) Mean speed quantification (e) and bacterial tracks of cells in control versus Van@GO treated samples (f-g). (h) SOD activity measurement in different samples. In this context, next we investigated the oxidative stress related expression of super oxide dismutase (SOD) in control and treated VRSA cultures. As shown in figure 5h, there is significant upregulation of SOD in Van@GO samples in comparison to control bacterial cells (student t-test, *p-value = 0.01) indicating that exposure of Van@GO to VRSA, synergistically improves the efficacy of Vancomycin via oxidative stress, producing free radicals. Enhanced turnover of SOD is an imperative line of defense in bacterial cells against postponement in development and survival against ROS.56 Steady and controlled elution of the antibiotics or therapeutic molecules from nanocarrier further potentiates antibacterial effect. Degrees of


20


Page 22 of 31

freedom regarding chain rotation of the therapeutic cargo on nanocarrier also influences therapeutic outcome.49 Further, we verified this notion using commercial ROS-specific probes (kit) to cross check the free radical production (supporting fig S3). This clearly indicates the Van@GO combinations triggers functional response in VRSA to inhibit growth, and colonization in biofilm and thus, enhancing propensity of Vancomycin.

4. Conclusion We have described a new class of polyvalent inhibition strategy of VRSA by combining the synergistic effect of GO and Vancomycin in Van@GO. High 2D surface area of GO and inherent negative charges provide a conducive environment towards the high loading of Vancomycin molecules through stable interactions. In Van@GO, the GO enhances the hydrophilicity of Vancomycin, which in turn, increases the interactions of vancomycin to bacterial cell leading to increased therapeutic efficacy. Thus, the present study provides an alternative approach based on GO to meet the existing challenges of resistant bacteria. If the drug molecules in proximity of a bacterium are more, the antibacterial propensity of Van@GO may be further enhanced. Associated content Supporting information The supporting information is available free of charge on the ACS publication website. Time lapse of untreated VRSA as a positive control demonstrate the random motion of S. aureus. Time lapse of Van@GO treated VRSA demonstrate the motion of very few S. aureus cells due to induced ROS based toxicity by the synergistic influence of antibiotic due inorganic metal


21

Page 23 of 31 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


composite. SEM micrograph of GO and Van@GO. Effect of varying concentration of GO loaded Vancomycin on bacterial density. Quantification of ROS and free radical production. 5. Acknowledgements The authors acknowledge the monetary funding of CAS program of the Departments of Zoology, and Physics for the consummation of the present work. Biophysical laboratory of the Department of Physics of Banaras Hindu University, Varanasi, India is also acknowledged for characterization facility. Authors also acknowledge Dr. Gopal Nath, Department of Microbiology, Institute of Medicine Science, Banaras Hindu University, Varanasi, India for the generous gift in the form of S. aureus strain. We thanks Dr Vaibhav Pandit, Dynex Technologies for the proof reading and language corrections. Author Contributions The conceptualization of the current work was by Dr. Vinod Kumar. All experiments were done by Vimal Singh, and Vinod Kumar. The formal analysis of the data was done by Ms. Sunayana Kashyap, Vimal Kishore, and Ajay Vikram Singh. The work was conducted under the supervision of Metin Sitti, Prof. Preeti S Saxena and Prof. Anchal Srivastava. The manuscript was composed through the commitments of all authors. All the authors have offered endorsement to the last form of the manuscript. Conflicts of Interest The authors declare that there are no conflicts of interest. References (1)

Fair, R. J.; Tor, Y. Antibiotics and Bacterial Resistance in the 21st Century. Perspect.


22


Page 24 of 31

Medicin. Chem. 2014, 6, 25–64. (2)

Klevens, R. M.; Edwards, J. R.; Richards, C. L.; Horan, T. C.; Gaynes, R. P.; Pollock, D. A.; Cardo, D. M. Estimating Health Care-Associated Infections and Deaths in U.S. Hospitals, 2002; 2007; Vol. 122.

(3)

Wang, L.; Chen, Y. P.; Miller, K. P.; Cash, B. M.; Jones, S.; Glenn, S.; Benicewicz, B. C.; Decho, A. W. Functionalised Nanoparticles Complexed with Antibiotic Efficiently Kill MRSA and Other Bacteria. Chem. Commun. (Camb). 2014, 50 (81), 12030–12033.

(4)

Klevens, R. M.; Morrison, M. A.; Nadle, J.; Petit, S.; Gershman, K.; Ray, S.; Harrison, L. H.; Lynfield, R.; Dumyati, G.; Townes, J. M.; Craig, A. S.; Zell, E. R.; Fosheim, G. E.; McDougal, L.K.; Carey, R. B.; Fridkin, S. K. Invasive Methicillin-Resistant Staphylococcus Aureus Infections in the United States. JAMA 2007, 298 (15), 1763–1771.

(5)

Kaur, D. C.; Chate, S. S. Study of Antibiotic Resistance Pattern in Methicillin Resistant Staphylococcus Aureus with Special Reference to Newer Antibiotic. J. Glob. Infect. Dis. 2015, 7 (2), 78–84.

(6)

Chakraborty, S. P.; Sahu, S. K.; Pramanik, P.; Roy, S. In Vitro Antimicrobial Activity of Nanoconjugated Vancomycin against Drug Resistant Staphylococcus Aureus. Int. J. Pharm. 2012, 436, 659–676.

(7)

Ventola, C. L. The Antibiotic Resistance Crisis: Part 1: Causes and Threats. Pharm. Ther. 2015, 40 (4), 277–283.

(8)

Cheng, J.; Meziani, M. J.; Sun, Y.-P.; Cheng, S. H. Poly(Ethylene Glycol)-Conjugated Multi-Walled Carbon Nanotubes as an Efficient Drug Carrier for Overcoming Multidrug Resistance. Toxicol. Appl. Pharmacol. 2011, 250 (2), 184–193.


23

Page 25 of 31 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


(9)

Li, X.; Robinson, S. M.; Gupta, A.; Saha, K.; Jiang, Z.; Moyano, D. F.; Sahar, A.; Riley, M. A.; Rotello, V. M. Functional Gold Nanoparticles as Potent Antimicrobial Agents against Multi-Drug-Resistant Bacteria. ACS Nano 2014, 8 (10), 10682–10686.

(10)

Choi, S. K.; Myc, A.; Silpe, J. E.; Sumit, M.; Wong, P. T.; Mccarthy, K.; Desai, A. M.; Thomas, T. P.; Kotlyar, A.; Holl, M. M. B.; Orr, B. G.; Baker, J. R. Dendrimer-Based Multivalent Vancomycin Nanoplatform for Targeting the Drug-Resistant Bacterial Surface. ACS Nano 2013, 7 (1), 214–228.

(11)

Mohammed Fayaz, A.; Girilal, M.; Mahdy, S. A.; Somsundar, S. S.; Venkatesan, R.; Kalaichelvan, P. T. Vancomycin Bound Biogenic Gold Nanoparticles: A Different Perspective for Development of Anti VRSA Agents. Process Biochem. 2011, 46 (3), 636– 641.

(12)

Chakraborty, S. P.; Pramanik, P.; Roy, S. Protective Role of Nanostructure Vancomycin against Sensitive Staphylococcus Aureus Induced Oxidative Stress and DNA Damage. Int. J. Pharm. Sci. Res. 2012, 3 (2), 405–415.

(13)

Baptista, P. V.; McCusker, M. P.; Carvalho, A.; Ferreira, D. A.; Mohan, N. M.; Martins, M.; Fernandes, A. R. Nano-Strategies to Fight Multidrug Resistant Bacteria-"A Battle of the Titans". Front. Microbiol. 2018, 9 (JUL), 1–26.

(14)

Li, Y.; Zhao, Z.; Zhang, J.; Kwok, R. T. K.; Xie, S.; Tang, R.; Jia, Y.; Yang, J.; Wang, L.; Lam, J. W. Y.; Zheng,, W.; Jiang, X.;Tang, B. Z. A Bifunctional Aggregation-Induced Emission Luminogen for Monitoring and Killing of Multidrug-Resistant Bacteria. Adv. Funct. Mater. 2018, 28 (42), 1804632.

(15)

Feng, Q. L.; Wu, J.; Chen, G. Q.; Cui, F. Z.; Kim, T. N.; Kim, J. O. A Mechanistic Study


24


Page 26 of 31

of the Antibacterial Effect of Silver Ions OnEscherichia Coli AndStaphylococcus Aureus. J. Biomed. Mater. Res. 2000, 52 (4), 662–668. (16)

Das, B.; Dash, S. K.; Mandal, D.; Ghosh, T.; Chattopadhyay, S.; Tripathy, S.; Das, S.; Dey, S. K.; Das, D.; Roy, S. Green Synthesized Silver Nanoparticles Destroy Multidrug Resistant Bacteria via Reactive Oxygen Species Mediated Membrane Damage. Arab. J. Chem. 2017, 10 (6), 862–876.

(17)

Cavassin, E. D.; Francisco, L.; Figueiredo, P. De; Otoch, J. P.; Seckler, M. M.; Oliveira, R. A. De; Franco, F. F.; Marangoni, V. S.; Zucolotto, V.; Levin, A. S. S; Costa, S. F. Comparison of Methods to Detect the in Vitro Activity of Silver Nanoparticles (AgNP) against Multidrug Resistant Bacteria. J. Nanobiotechnology 2015, 13, 64.

(18)

Matai, I.; Sachdev, A.; Dubey, P.; Uday Kumar, S.; Bhushan, B.; Gopinath, P. Antibacterial Activity and Mechanism of Ag-ZnO Nanocomposite on S. Aureus and GFPExpressing Antibiotic Resistant E. Coli. Colloids and Surfaces B:Biointerfaces 2014, 115, 359–367.

(19)

Arakha, M.; Saleem, M.; Mallick, B. C.; Jha, S. The Effects of Interfacial Potential on Antimicrobial Propensity of ZnO Nanoparticle. Sci. Rep. 2015, 5, 9578.

(20)

Shende, S.; Ingle, A. P.; Gade, A.; Rai, M. Green Synthesis of Copper Nanoparticles by Citrus Medica Linn. (Idilimbu) Juice and Its Antimicrobial Activity. World J. Microbiol. Biotechnol. 2015, 31 (6), 865–873.

(21)

Kuo, W. S.; Shao, Y. T.; Huang, K. S.; Chou, T. M.; Yang, C. H. Antimicrobial AminoFunctionalized Nitrogen-Doped Graphene Quantum Dots for Eliminating MultidrugResistant Species in Dual-Modality Photodynamic Therapy and Bioimaging under Two-


25

Page 27 of 31 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


Photon Excitation. ACS Appl. Mater. Interfaces 2018, 10 (17), 14438–14446. (22)

Marambio-Jones, C.; Hoek, E. M. V. A Review of the Antibacterial Effects of Silver Nanomaterials and Potential Implications for Human Health and the Environment. J. Nanoparticle Res. 2010, 12, 1531–1551.

(23)

Panáček, A.; Kvítek, L.; Smékalová, M.; Večeřová, R.; Kolář, M.; Röderová, M.; Dyčka, F.; Šebela, M.; Prucek, R.; Tomanec, O.; Zboril, R. Bacterial Resistance to Silver Nanoparticles and How to Overcome It. Nat. Nanotechnol. 2018, 13 (1), 65–71.

(24)

Kalan, L. R.; Pepin, D. M.; Ul-Haq, I.; Miller, S. B.; Hay, M. E.; Precht, R. J. Targeting Biofilms of Multidrug-Resistant Bacteria with Silver Oxynitrate. Int. J. Antimicrob. Agents 2017, 49 (6), 719–726.

(25)

Shanmuganathan, R.; MubarakAli, D.; Prabakar, D.; Muthukumar, H.; Thajuddin, N.; Kumar, S. S.; Pugazhendhi, A. An Enhancement of Antimicrobial Efficacy of Biogenic and Ceftriaxone-Conjugated Silver Nanoparticles: Green Approach. Environ. Sci. Pollut. Res. 2018, 25 (11), 10362–10370.

(26)

Georgakilas, V.; Tiwari, J. N.; Kemp, K. C.; Perman, J. A.; Bourlinos, A. B.; Kim, K. S.; Zboril, R. Noncovalent Functionalization of Graphene and Graphene Oxide for Energy Materials, Biosensing, Catalytic, and Biomedical Applications. Chem. Rev. 2016, 116, 5464–5519.

(27)

Wang, Y.; Li, Z.; Wang, J.; Li, J.; Lin, Y. Graphene and Graphene Oxide: Biofunctionalization and Applications in Biotechnology. Trends Biotechnol. 2011, 29 (5), 205–212.

(28)

Huang, X.; Qi, X.; Boey, F.; Zhang, H. Graphene-Based Composites. Chem. Soc. Rev.


26


Page 28 of 31

2012, 41 (2), 666–686. (29)

Wang, Y.; Shao, Y.; Matson, D. W.; Li, J.; Lin, Y. Nitrogen-Doped Graphene and Its Application in Electrochemical Biosensing. ACS Nano 2010, 4 (4), 1790–1798.

(30)

Sun, X.; Liu, Z.; Welsher, K.; Robinson, J. T.; Goodwin, A.; Zaric, S.; Dai, H. NanoGraphene Oxide for Cellular Imaging and Drug Delivery. Nano Res. 2008, 1 (3), 203–212.

(31)

Kim, H.; Namgung, R.; Singha, K.; Oh, I.-K.; Kim, W. J. Graphene OxidePolyethylenimine Nanoconstruct as a Gene Delivery Vector and Bioimaging Tool. Bioconjug. Chem. 2011, 22 (12), 2558–2567.

(32)

Gurunathan, S.; Han, J. W.; Dayem, A. A.; Eppakayala, V.; Kim, J.-H. Oxidative StressMediated Antibacterial Activity of Graphene Oxide and Reduced Graphene Oxide in Pseudomonas Aeruginosa. Int. J. Nanomedicine 2012, 7, 5901–5914.

(33)

Li, C.; Wang, X.; Chen, F.; Zhang, C.; Zhi, X.; Wang, K.; Cui, D. The Antifungal Activity of Graphene Oxide Silver Nanocomposites. Biomaterials 2013, 34 (15), 3882–3890.

(34)

Watanakunakorn, C. Mode of Action and In-Vitro Activity of Vancomycin. J. Antimicrob. Chemother. 1984, 14, 7–18.

(35)

Sinha Roy, R.; Yang, P.; Kodali, S.; Xiong, Y.; Kim, R. M.; Griffin, P. R.; Onishi, H. R.; Kohler, J.; Silver, L. L.; Chapman, K. Direct Interaction of a Vancomycin Derivative with Bacterial Enzymes Involved in Cell Wall Biosynthesis. Chem. Biol. 2001, 8 (11), 1095– 1106.

(36)

Marcano, D. C.; Kosynkin, D. V; Berlin, J. M.; Sinitskii, A.; Sun, Z.; Slesarev, A.; Alemany, L. B.; Lu, W.; Tour, J. M. Improved Synthesis of Graphene Oxide. ACS Nano 2010, 4 (8), 4806–4814.


27

Page 29 of 31 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


(37)

Gu, H.; Ho, P. L.; Tong, E.; Wang, L.; Xu, B. Presenting Vancomycin on Nanoparticles to Enhance Antimicrobial Activities. Nano Lett. 2003, 3 (9), 1261–1263.

(38)

Jijie, R.; Barras, A.; Bouckaert, J.; Dumitrascu, N.; Szunerits, S.; Boukherroub, R. Enhanced Antibacterial Activity of Carbon Dots Functionalized with Ampicillin Combined with Visible Light Triggered Photodynamic Effects. Colloids Surfaces B Biointerfaces 2018, 170 (April), 347–354.

(39)

Singh, A. V.; Vyas, V.; Patil, R.; Sharma, V.; Scopelliti, P. E.; Bongiorno, G.; Podestà, A.; Lenardi, C.; Gade, W. N.; Milani, P. Quantitative Characterization of the Influence of the Nanoscale Morphology of Nanostructured Surfaces on Bacterial Adhesion and Biofilm Formation. PLoS One 2011, 6 (9), e25029.

(40)

Srinivasan, A.; Dick, J. D.; Perl, T. M. Vancomycin Resistance in Staphylococci. Clin. Microbiol. Rev. 2002, 15 (3), 430–438.

(41)

Chang, S.; Sievert, D. M.; Hageman, J. C.; Boulton, M. L.; Tenover, F. C.; Downes, F. P.; Shah, S.; Rudrik, J. T.; Pupp, G. R.; Brown, W. J.; Cardo, D.; Fridkin, S. K. Infection with Vancomycin-Resistant Staphylococcus Aureus Containing the VanA Resistance Gene. N. Engl. J. Med. 2003, 348, 1342–1347.

(42)

Guo, H.-L.; Wang, X.-F.; Qian, Q.-Y.; Wang, F.-B.; Xia, X.-H. A Green Approach to the Synthesis of Graphene Nanosheets. ACS Nano 2009, 3 (9), 2653–2659.

(43)

Ferrari, A. C.; Meyer, J. C.; Scardaci, V.; Casiraghi, C.; Lazzeri, M.; Mauri, F.; Piscanec, S.; Jiang, D.; Novoselov, K. S.; Roth, S.; Geim A. K. Raman Spectrum of Graphene and Graphene Layers. Phys. Rev. Lett. 2006, 187401, 1–4.

(44)

Pimenta, M. A.; Dresselhaus, G.; Dresselhaus, M. S.; Canc, L. G. Studying Disorder in


28


Page 30 of 31

Graphite-Based Systems by Raman Spectroscopy. Phys. Chem. Chem. Phys. 2007, 9, 1276–1291. (45)

Ferrari, A. C.; Robertson, J. Interpretation of Raman Spectra of Disordered and Amorphous Carbon. Phys. Rev. B 2000, 61 (20), 95–107.

(46)

Paredes, J. I.; S, V.-R.; Martinez-Alonso, A.; Tascon, J. M. D. Graphene Oxide Dispersions in Organic Solvents. Langmuir 2008, 24, 10560–10564.

(47)

Aboutalebi, S. H.; Gudarzi, M. M.; Zheng, Q. Bin; Kim, J.-K. Spontaneous Formation of Liquid Crystals in Ultralarge Graphene Oxide Dispersions. Adv. Funct. Mater. 2011, 21, 2978–2988.

(48)

Singh, A. V.; Jahnke, T.; Kishore, V.; Park, B.-W.; Batuwangala, M.; Bill, J.; Sitti, M. Cancer Cells Biomineralize Ionic Gold into Nanoparticles-Microplates via Secreting Defense Proteins with Specific Gold-Binding Peptides. Acta Biomater. 2018, 71, 61–71.

(49)

Singh, A. V.; Alapan, Y.; Jahnke, T.; Laux, P.; Luch, A.; Aghakhani, A.; Kharratian, S.; Onbasli, M. C.; Bill, J.; Sitti, M. Seed-Mediated Synthesis of Plasmonic Gold Nanoribbons Using Cancer Cells for Hyperthermia Applications. J. Mater. Chem. B 2018, 6 (46), 7573–7581.

(50)

Singh, A. V.; Jahnke, T.; Wang, S.; Xiao, Y.; Alapan, Y.; Kharratian, S.; Onbasli, M. C.; Kozielski, K.; David, H.; Richter, G.; Bill, J.; Laux, P.; Luch, A.; Sitti, M. Anisotropic Gold Nanostructures: Optimization via in Silico Modeling for Hyperthermia. ACS Appl. Nano Mater. 2018, 1 (11), 6205–6216.

(51)

Krishnamoorthy, K.; Veerapandian, M.; Yun, K.; Kim, S. The Chemical and Structural Analysis of Graphene Oxide with Different Degrees of Oxidation. Carbon N. Y. 2012, 53,


29

Page 31 of 31 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


38–49. (52)

Chen, J.; Yao, B.; Li, C.; Shi, G. An Improved Hummers Method for Eco-Friendly Synthesis of Graphene Oxide. Carbon N. Y. 2013, 64 (1), 225–229.

(53)

Paiva, C. N.; Bozza, M. T. Are Reactive Oxygen Species Always Detrimental to Pathogens? Antioxid. Redox Signal. 2014, 20 (6), 1000–1037.

(54)

Singh, A. V.; Mehta, K. K.; Worley, K.; Dordick, J. S.; Kane, R. S.; Wan, L. Q. Carbon Nanotube-Induced Loss of Multicellular Chirality on Micropatterned Substrate Is Mediated by Oxidative Stress. ACS Nano 2014, 8 (3), 2196–2205.

(55)

Singh, A. V.; Vyas, V.; Salve, T. S.; Cortelli, D.; Dellasega, D.; Podestà, A.; Milani, P.; Gade, W. N. Biofilm Formation on Nanostructured Titanium Oxide Surfaces and a Micro/Nanofabrication-Based

Preventive

Strategy

Using

Colloidal

Lithography.

Biofabrication 2012, 4 (2), 025001. (56)

Poole, K. Mechanisms of Bacterial Biocide and Antibiotic Resistance. J. Appl. Microbiol. 2002, 92 Suppl, 55S–64S.


30

Graphene oxide synergistically enhances antibiotic efficacy in

Recommend Documents