"Improved Bactericidal Activity of Polyethylenimine Grafted Graphene Oxide Nanocomposite against Staphylococcus aureus and Escherichia coli"

Global emergence of pathogenic bacteria is becoming a major concern to humanâ€™s health because of the resistance of bacteria towards antibiotics overuse...

Graphene is the 1 st 2-dimensional (2D), single atomic sheet of graphite, hexagonally arranged lattice structure of sp 2 hybridized carbon atoms [7,18,19]. Likewise, Graphene oxide (GO) is the derivatives of graphene with sp 3 hybridization of carbon atoms, highly oxidative, obtained by chemical oxidation and subsequently exfoliation [20], hydrophilic in nature, possessing a large number of oxygen-bearing functional groups including epoxy and hydroxyl on the basal plane, and carboxyl groups on the edges [21,22]. These oxygen-containing functional groups assist the chemical functionalization of GO sheet with other molecules [22].
Various studies on the antibacterial effect of GO has reported its bactericidal property through somatic damage and oxidative stress [17,27,28]. Along with physical damage [27], oxidative mechanism also plays its part in deactivating bacterial cell by reactive oxygen species (ROS) production in GO [26,29].
Liu et al. have studied the antibacterial effect of four different graphene-based material (GO, Gt, Gr, rGO) and found GO with higher bactericidal property comparing to others [30]. In another study done by Barbolina et al. observed no antibacterial effect of pure GO against Gram-positive S. aureus and Gram-negative E. coli [31]. Furthermore, other studies on the functionalization of GO with various nanoparticles and polymers has been reported to improve its antibacterial property and stabilization [32,33]. Polyethylenimine (PEI) is polycationic synthetic polymer prepared from aziridine by polymerization, hydrophilic, branched or unbranched, containing primary, secondary and tertiary amino group (-NH 2 ) in its structure, that can be protonated [34,35]. Because of being highly positive charge, high membrane permeability and exceptional transferability, PEI is extensively used in gene transfer and drug delivery [34,36]. PEI itself is considered as microbicidal agent, and it is also used as an enhancer in the field of microbiology to strength the antibacterial property of both hydrophilic and hydrophobic antibiotic [37]. Gao et al. prepared quaternate Polyethylenimine (QPEI), that exhibited excellent antibacterial activity, associated with the degree of cationic charge on its backbone chain [38]. A study by Azevedo et al. used nanoPEI to inhibit bacterial growth and concluded that the antibacterial property of PEI mainly depends on high concentration as well as species specificity [39]. PEI can be linked to GO via nucleophilic addition reaction between the PEI amine group and carboxyl and epoxy group of graphene oxide [40,41]. Here in, keeping in view the antibacterial property of both the materials, we have prepared PEI grafted graphene oxide (PEI-GO) nanocomposite through the functionalization of PEI on the graphene sheet via C-N bond. The schematic illustration is presented in Figure 1. The nanocomposite was observed through various characterization techniques. The antibacterial activity of as produced PEI-GO was conducted against two bacterial strains (S. aureus and E. coli as the representative bacteria), besides that time dynamic bactericidal activity was also studied. It is expected that our nanocomposite will have excellent biocidal activity because of the synergy effect of PEI and GO.

Preparation of PEI-GO Nanocomposite
Graphene oxide was grafted with PEI according to the previously reported process with little adjustment [42]. 20 mg of graphene oxide was ultrasonicated in 10 mL of ultrapure water for 2 hours to get a homogeneous dispersion. Then 100 mg PEI was added and sonicated for an additional 30 minutes. After that, 60 mg EDC and 30 mg NHS were added gradually in the dispersion and sonicated for a further 15 minutes. Next, the material in the flask was shifted on the magnetic stirrer for one day, and speed of the stirrer was adjusted to 850 rpm. The whole procedure was performed at ambient temperature. The composite was obtained by centrifugation followed by three washing with ultrapure water and subsequently dried in the oven at 50 o C.

Characterization
Transmission electron microscopy (TEM) was performed at an accelerating voltage of 200 kV on a JEOL model 2100 HR instrument (TEM, JEOL, Ltd., Japan). Shimadzu UV-1800 spectrophotometer was used to carry out the UV−vis absorption spectra in the range of 200 to 800 for nanocomposite (Shimadzu, Japan). Fouriertransform infrared spectrophotometer (FTIR) was used to obtain FTIR spectra on Nicolet Nexus 470 FTIR Spectrophotometer (Thermo Electron co., USA) at a wavelength of 500 to 4000 cm -1 . The X−ray diffraction patterns were taken on a Bruker D8 advance X−ray diffractometer with Cu-Kα X-ray radiation (λ=1.5406 Å) with 40 kV voltage at the 2 o /minute scanning rate (Bruker AXS Ltd., Germany).
Raman spectra were measured using LABRAM HR 800 microscopy confocal Raman spectrometer using 532 nm excitation wavelength with x50 objective (HORIBA Corporation, Japan). Nanocomposite zeta potential was measured using zeta potential instrument by diluting the sample in water (Malvern zeta-sizer nano ZS). NaCl) to remove extra residual macromolecules [17]. Optical density (OD 600 nm ) was measured by spectrophotometer after resuspending the pellets in saline solution and diluted to 10 6 CFU/mL.

Antibacterial Test
For antibacterial assay, 10 μL of bacterial suspensions were mixed in 900 μL of saline solution comprising a different concentration of GO, PEI, PEI-GO. Tubes containing the above suspensions were transferred into incubator shaker for 5 hours at 37 o C with shaking speed 200 rpm. Afterwards, 100 μL of suspension from the above samples were spread evenly on the LB agar plates, and all the plates were incubated overnight at 37 o C [43]. The same procedure was adopted for the control sample without any material treatment. Bacterial colonies were counted on LB agar plates and survival percentage was calculated by compairing control group.
All experiments were repeated three times.

Minimum Inhibitory Concentration (MIC)
Minimum inhibitory concentration of GO, PEI, PEI-GO were ascertained by the previously reported microdilution technique [44] with little modification. In brief, log phase S. aureus and E. coli  Figure 3a, the spectrum of bare GO indicates two representative peaks, the sharp peak is observed at bonds, and the shoulder peak is noticed at 300 nm link to the n-π* transitions of C=O bonds [45]. For PEI, a sharp absorption peak is observed at 200 nm. On the other hand, the spectrum of PEI-GO is observed same as GO with a fresh absorption peak at 200 nm, witness the conjugation of PEI with GO. The FTIR spectrophotometer was used to confirm the formation of PEI-GO complex (Figure 3b). In the bare GO spectrum, the main absorption peak at 3424 cm -1 is ascribed to -OH stretching vibration. The peak at 1714 cm -1 shows the Stretching vibration of carboxyl functional group of GO. Whereas the band 1624 cm -1 is considered as C=C aromatic Stretching, that at 1224 cm -1 and 1063 cm -1 are assigned to epoxy C-O stretching vibration [46].
The Infrared spectrum of PEI indicates absorption peak at 1573 cm -1 , and 1111 cm -1 are linked to the bend vibration of N-H bond of primary and secondary amine groups, respectively [38]. After PEI and GO reaction, downshift in a peak at 3418 cm -1 is observed assign to hydroxyl stretching, and two other peaks are gained at 2916 and 2855 cm -1 , ascribed to the symmetric and asymmetric stretching mode of PEI methylene groups [47]. The peak at 1714 cm -1 is hardly noticeable, and a new strong peak is detected at 1608 cm -1 indicate the stretching mode of amide bond because of the reaction of polyethylenimine -NH 2 groups with graphene oxide -COOH groups. Furthermore, disappearance of peak at 1224 cm -1 , and the prominent peak at 1067 cm -1 also indicate the formation of C-N bond via epoxy groups of GO and amine groups of PEIs.
XRD measurements were performed to determine the changes in the crystalline structure of GO before, and after functionalization with PEI. As presented in Figure 3c, the XRD pattern of bare GO expresses one characteristic peak at 2θ = 11.2 o corresponding to the (001) crystalline plane having an interlayer-spacing 0.793 nm.
This interlayer-spacing is because of the oxygen-bearing functional groups of GO present in the layers [12]. After PEI conjugation with GO, two peaks are observed, the broad peak at 2θ = 20.2 o might be assigned to the non-crystalline diffraction of PEI, and the peak at

Antibacterial Activity
The coli, S. aureus shows nearly complete inhibition at 20 μg/mL, which can be seen in Figure 5a. The difference in bacterial reduction could be attributed to more resistance of E. coli as compared to S. aureus.
Although S. aureus (Gram-positive bacteria) contain a thick layer of peptidoglycan in its membrane (thickness range 20 -80 nm), but it lacks an outer membrane. In contrast, E. coli (Gram-negative bacteria) encompasses a thin layer of peptidoglycan (thickness range 7 -8 nm), but it keeps another additional layer known as the outer membrane in its structure, this outer membrane of E. coli express resistance to the direct contact of bacteria with nanocomposite [27,50,51].
Moreover, PEI exhibit significantly higher bacterial inhibition rate towards Gram-positive bacteria [35], this could be attributed to greater cationic charge transfer onto the negative charge membrane, which increases the cell permeability [52]

Possible Bactericidal Phenomena
The bactericidal property is attributed to the damage caused

Conclusion
In the present study, we have synthesized Polyethylenimine

Competing Interest
The authors declare no competing interest. a b