Graphene Oxide-Lignin/Silk Fibroin/ZnO Nanobiocomposite: A Novel Bioactive Scaffold With Antibacterial Activity

In this study, a novel nanobiocomposite was synthesized using graphene oxide, lignin, silk broin and ZnO and used in biological elds. To synthesize this structure, after preparing graphene oxide by the Hummer method, lignin, silk broin, and ZnO nanoparticles (NPs) were added to it, respectively. Also, ZnO NPs with a particle size of about 18 nm to 33 nm was synthesized via Camellia sinensis extract by green methodology. The synthesized structure was examined as anti-biolm agent and it was observed that the Graphene oxide-lignin/silk broin/ZnO nanobiocomposite has a signicant ability to prevent the formation of P. aeruginosa biolm. In addition, due to the importance of the possibility of using this structure in biological environments, its toxicity and blood compatibility were also evaluated. According to the obtained results from MTT assay, the viability percentages of Hu02 cells treated with Graphene oxide-lignin/silk broin/ZnO nanobiocomposite after 24, 48, and 72 h of incubation were 89.96%, 89.32%, and 91.28%. On the other hand, the hemolysis percentage of the synthesized structure after 24 h and 72 h of extraction was 9.5% and 11.76% respectively. As a result, the synthesized structure is hemocompatible and had no toxic effects on Hu02 cells.

should be noted that reports of graphene oxide toxicity have limited its use in biological applications alone and usually utilized in combination with safe material as a nanobiocomposite [26]. In this regard, various natural biorenewable resources such as starch, chitin, chitosan, cellulose, alginate, hyaluronic acid, gelatin, collagen, silk broin, and lignin have been used [1]. Lignin is an organic biopolymer with a cross-linked polyphenolic structure and it is found as support tissue in the vast majority of the plants [27,28]. Lignin has received considerable attention in health care due to its non-toxicity and biocompatibility.
Its use has been studied as antioxidant, antimicrobial, anti-tumor, and antiviral agents [29]. It can also be utilized in elds of antidiabetic materials, drug delivery and, tissue engineering [29]. In addition to lignin, silk broin as an important biomaterial is a major component of silk protein with polypeptide chains with a molecular weight of 200 to 350 kDa [30]. Silk broin with properties such as biocompatibility, blood compatibility, non-carcinogenicity, non-toxicity, and suitable mechanical properties has been highly regarded by researchers [17]. Based on previous studies, the combination of silk broin with materials such as graphene oxide, natural polymers, and metal/metal oxide/metal hydroxide NPs enhances the mechanical and antibacterial properties of a composite made from silk broin [17,[31][32][33][34]. Metal NPs play an important role in a variety of biological applications, including anti-microbial and anti-bacterial materials, biosensing, drug delivery, bio-imaging, and etc. [4,[35][36][37][38]. There are various methods for the synthesis of metal oxide NPs, which can be referred to mechanical milling, laser ablation, ion sputtering, physical vapor deposition, chemical vapor deposition, sol-gel, chemical reduction, hydrothermal, solvothermal, spray pyrolysis, laser pyrolysis, and ame pyrolysis [39]. These methods also have drawbacks such as need the large amounts of energy, long reaction time, high cost, low e ciency of NPs production, use of toxic and corrosive substances, di cult reaction conditions, and production of impurities [39]. The mentioned negative points caused the introduction of alternative methods. Green synthesis of metal/metal oxide NPs by plant extract has been highly regarded as a new method in recent years. During this method, the plant extract can act as a reducing and stabilizing agent and convert metal ions into metal NPs [40,41]. Herein, a novel nanobiocomposite base on graphene oxide, lignin, silk broin, and ZnO was synthesized and its application as a substance with antibio lm properties was evaluated.
Also, due to the importance of the possibility of using this substance in biological environments, its toxicity and blood compatibility were investigated and it was observed that this new antibio lm substance is not signi cantly toxic and is also compatible with blood.

2-1-General
In this study, all reagents, chemical materials, and solvents were purchased from Merck and Flucka except the silkworm cocoons that are taken from local stores. Also, the 14,000 Da dialysis tubing cellulose membrane was purchased from Sigma-Aldrich Company. The structure of synthesized nanobiocomposite was evaluated from different points of view by using Fourier-transform infrared (FT-IR) spectroscopy, X-Ray Diffraction (XRD) Analysis, Thermogravimetric Analysis (TGA), Energy Dispersive X-Ray (EDX) Analysis, and Field-Emission Scanning Electron Microscopy (FE-SEM). FT-IR analysis was performed using AVATAR Thermo device in the range of 450 cm −1 to 4500 cm −1 and using the potassium bromide pellets method. EDX and FE-SEM analysis was carried out using EM8000 KYKY apparatus and ZEISS SIGMA VP model, respectively. XRD analysis was evaluated using PANalytical X-PERT-PRO MPD at 2θ, 5° to 90°. TGA was performed using STA504 analyzer in a temperature range of 50°C to 550°C with a temperature rate of 10°C/min in air.

2-2-Preparation of graphene oxide
Graphene oxide was prepared using the modi ed Hummer method [42]. In this regard, 1 g of graphite and 23 ml of sulfuric acid (98 %) were poured into a 1 liter beaker and they were mixed for 5 minutes. Then, 0.5 g of sodium nitrate was added to the blend and they were mixed for 20 minutes at 65°C. The beaker was then placed in an ultrasonic bath at room temperature for 20 minutes to completely dissolve the components. Afterwards, 3.5 g of potassium permanganate was added to the mixture during one hour until a sludge-like substance formed. This process was performed while the components were mixing in an ultrasonic bath with plenty of ice particles. In the next step, mixture was kept for further 30 minutes in an ultrasonic bath (25°C) to complete the reaction and subsequently, 50 ml of distilled water was added to the beaker and mixed for 30 minutes at 98°C. 700 ml of distilled water and 12 ml of hydrogen peroxide were added to the obtained mixture to observed a signi cant amount of foam. The pH of the mixture was then set using a 2% HCl solution (2 ml HCl in 100 ml distilled water). The mixture was remained stationary for one day and after the precipitate settles. After the mentioned time, the containers water was changed and elution process was repeated for 3 times. Finally, the precipitate was placed in an oven at 60°C for 24 hours to dry (Fig. 1).

2-3-Extraction of silk broin
Extraction of silk broin was performed using the method reported in literatures [22,43]. Initially, three silk worm cocoons with a perfectly clean appearance were divided into small pieces and allowed to boil in 0.21% w/v sodium carbonate aqueous solution for 2 hours. After the mentioned time, the bers were separated and washed 6 times with distilled water. The laments should be well separated from each other's during washing so that the pollutants are washed well. After washing, the bers were dried at room temperature for 12 hours. The dried bers were weighed (0.644) and then, 9.3 M lithium bromide solution was made by using 6.44 g of water (10 times of the dry bers weight). Then, to remove the remaining lithium bromide, the obtained solution enters the dialysis cellulose membrane and is placed in the presence of distilled water, and this process continues for 3 days at room temperature. Finally, the obtained silk broin was stored at -4°C for later use.

2-4-Preparation of NPs 2-4-1-Preparation of Camellia sinensis extract
First, 10 g of dried leaves of Camellia sinensis were mixed with 200 ml of deionized water and heated at 70°C for 10 minutes. The resulting solution was then passed through lter paper after cooling.
2-4-2-Preparation of ZnO NPs 0.11 g Zn(CH CO ) .2H O and 25 ml of deionized water mixed with a magnetic stirrer for 30 minutes.
Then 1 ml of the extract was added to the container and it was stirred for 2 hours. In the next step, the pH of the solution was adjusted to 12 by using the 0.02 M NaOH solution. After the yellow color was observed, the solution was stirred vigorously for 3 hours. The obtained NPs were separated by using a centrifuge (10,000 r/min) and after washing with distilled water, they were dried in an oven at 50°C for 12 hours.
2-5-Preparation of Graphene oxide-lignin 0.4 g of graphene oxide and 45 ml of distilled water were mixed and placed in an ultrasonic bath for 30 min. Then 0.4 g of lignin was added to the previous mixture and the mixture was placed in an ultrasonic bath for another 30 minutes. The resulting mixture was stirred at 80°C for 12 hours. After this time, the reaction mixture was stored in the refrigerator for later use.

2-6-Preparation of Graphene oxide-lignin/silk broin/ZnO nanobiocomposite
At this stage, 10 ml of silk broin solution was added to 30 ml of Graphene oxide-lignin solution and re uxed for 12 hours. After the mentioned time, 0.03 g of zinc oxide powder was dispersed in 10 ml of distilled water and added to the previous blend. Subsequently the mixture was stirred for 12 hours under re ux conditions. Finally, the synthesized nanobiocomposite was freeze dried for 48 hours and stored in a cool and dry place.

2-7-MTT assay
First, the synthesized Graphene oxide-lignin/silk broin/ZnO nanobiocomposite was extracted. In this way, 50 mg of it was dispersed in 1 ml of PBS using shaker incubator for 24 h at 37°C [44]. Then, in order to measure the survival rate of Hu02 cell line (human skin broblast cells) in the vicinity of the Graphene oxide-lignin/silk broin/ZnO nanobiocomposite, MTT assay was performed. For this purpose, 1×10 5 cell/well was cultured in 96-well plates at optimal conditions (37°C, 5% CO 2 ) in humidi ed incubator.
Next, the growth media (10% FBS) was removed and the cells were washed twice with PBS. New maintenance RPMI (Roswell Park Memorial Institute) medium including nanobiocomposite extract was added and the cells were incubated for 24, 48, and 72 h. Also, attached RPMI without nanobiocomposite extract and cells in each well, were considered as negative control. Following, 10 µL solution of freshly prepared 5 mg/mL MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) in PBS was added to each well and allowed to incubate at 37°C for 4 h. Thereafter, the media with MTT solution was removed and 2-propanol was added at 100 µL/well. Next, the plates were shaken gently to facilitate formazan crystal solubilization [22]. The absorbance was measured at 590 nm using a microplate reader (STAT FAX 2100, BioTek, Winooski, USA). Finally, the percentage of cell toxicity and cell viability was calculated as follows [45]: (1) Viability % = 100 -Toxicity (2)

2-8-Hemolysis assay
This study was performed in accordance with the principles outlined in the Declaration of Helsinki. Also, the experimental methods and the procedure for taking informed satisfaction were approved by Semnan University of Medical Sciences, Ethics Research Committee. First, 50 mg of nanobiocomposite was dissolved in 1 ml PBS by shaker incubator at 37°C with two extraction time, 24 h and 72 h [44]. Next, hemolytic assay was performed to measure the potential lytic effects of the Graphene oxide-lignin/silk broin/ZnO nanobiocomposite on human red blood cells (RBCs). Then, Fresh blood sample was taken from a volunteer with the O negative blood type. A subsequent blood sample was diluted in PBS (1:20). After that, 100 µL of the solution was added in triplicate to 100 µL of each Graphene oxide-lignin/silk broin/ZnO nanobiocomposite extract (24 h and 72 h) in a 96-well plate. 1% Triton X-100 solution, which lyses 100% of RBCs and sterile 0.9% NaCl solution were also used as positive control and negative control, respectively. Then, the plate was incubated at 37°C for 1 h and samples were regained and centrifuged at 3000 rpm for 15 min [46]. The absorbance of each sample was measured by photometric analysis of supernatant at 414 nm using a microplate reader (STAT FAX 2100, BioTek, Winooski, USA).
Eventually, the hemolysis percentage of the samples was calculated using the following formula [47]:  Figure 2 shows the FT-IR spectrum of Graphene oxide-lignin and Graphene oxide-lignin/silk broin/ZnO nanobiocomposite. As shown in Figure 2(a), spectrum represents a mixture of graphene oxide and lignin.

3-1-1-FT-IR analysis
The broad peak observed in region 3421 cm −1 is related to the stretching vibration of the hydroxyl group of graphene oxide and lignin [22,31,49]. Peaks in the 1722 cm −1 and 1225 cm −1 regions may represent the stretching modes of carbonyl and C-O-C groups in the graphene oxide structure, respectively [22,50].
In addition, the presence of lignin is con rmed according to the peaks observed in areas 2927 cm −1 , 2840 cm −1 , and 1120 cm −1 [51]. These peaks are related to the stretching vibration of aliphatic C-H in methyl and methylene, symmetric stretching of CH 3 in methoxy, and stretching vibration mode of C-O in alcohol, respectively [51]. On the other hand, the peaks in the 1508 cm −1 and 1425 cm −1 indicate the vibrations of the aromatic skeleton in the lignin structure [51]. Figure 2(b) shows the structure of Graphene oxidelignin/silk broin/ZnO nanobiocomposite. Lignin and graphene oxide peaks are maintained in this spectrum. Also, the peak of ZnO NPs is observed in the region of about 500 cm −1 [52]. The presence of silk broin is also con rmed by observing peaks in 1519 cm −1 and 1535 cm −1 which are related to the N-H bending vibration of amide II [22]. In addition, the peak observed around 1650 cm −1 could demonstrate the presence of amides carbonyl in the structure of silk broin [22].

3-1-2-EDX analysis
The EDX spectrum of Graphene oxide-lignin/silk broin/ZnO nanobiocomposite can be seen in Fig. 3(a). As can be seen, all the expected elements in the nal structure are seen in the EDX image. The presence of zinc in the EDX spectrum con rms the presence of ZnO NPs. In addition, carbon and oxygen can be due to the presence of graphene oxide and lignin. Moreover, the presence of nitrogen is related to the amino acid structures of broin silk. In addition to examining the EDX spectrum, which was taken to con rm the elements of the structure, the distribution of the elements was also evaluated using elemental mapping pictures and as a result, it was observed that the elements have acceptable distribution ( Fig.  3(b)).

3-1-3-FE-SEM imaging
FE-SEM images were taken from Graphene oxide-lignin/silk broin/ZnO nanobiocomposite synthesis steps. As shown in Fig. 4(a), ZnO NPs were well synthesized with spherical shapes and a particle size of about 18 to 33 nm. Fig. 4(b) shows the morphology of the Graphene oxide-lignin composite and based on what is seen, the graphene oxide plates alongside with secondary structure (lignin) are clearly visible Fig.  4(c) and Fig. 4(d) shows Graphene oxide-lignin/silk broin and Graphene oxide-lignin/silk broin/ZnO respectively. Based on the FE-SEM image of the nal nanobiocomposite, ZnO NPs with the same size as Fig. 4(a) are well dispersed in the structure of Graphene oxide-lignin/silk broin/ZnO nanobiocomposite.

3-1-5-TG analysis
TGA analysis was performed to evaluate the correct formation and thermal stability of the nanobiocomposite (Fig. 5(e)). The rst mass reduction, which is in the temperature range of 50°C to 100°C, is related to the removal of trapped water, solvents and probable impurities from the Graphene oxide-lignin/silk broin/ZnO nanobiocomposite structure (about 20%) [17]. The second mass reduction occurs in the range of 150°C to 300 and during this process, about 30% of the mass was eliminated. Thermal degradation of lignin macromolecules occurs at temperatures around 150°C to 550°C [56]. In addition, a mass reduction in the range of 200°C to 300°C can be related to the pyrolysis of the oxygenated portions of graphene oxide, including the carboxyl, epoxide, and hydroxyl groups [22]. The third mass reduction occurs at 300°C to 500°C and 40% of the sample weight is lost. Mass reduction in the temperature range about 250°C to 400°C can be related to the destruction of peptide structures in silk broin [22]. In addition, ZnO NPs do not have a signi cant mass reduction of up to 500 degrees, and their partial mass reduction can be due to the release of absorbed moisture [57].

3-2-2-Hemocompatibility
The hemolysis percentage of the Graphene oxide-lignin/silk broin/ZnO nanobiocomposite after 24 h of extraction was 9.5%. This amount increased to 11.76% after 72 h of extraction. This is while 1% triton X-100 lysed almost all RBCs. Fig. 7 shows the histogram of hemolysis percentage, as well as image of 96well plate. It is also worth noting that these results are the average of three separate experiments. Based on the results, it can be said that Graphene oxide-lignin/silk broin/ZnO nanobiocomposite is hemocompatible.

3-2-3-Anti-bio lm activity
As shown in Fig. 8, the adsorption rate of polystyrene (as a positive control) at 570 nm was 0.84, which was reduced to 0.12 for Graphene oxide-lignin/silk broin/ZnO nanobiocomposite. In fact, the decrease in OD of the NB culture medium containing Graphene oxide-lignin/silk broin/ZnO nanobiocomposite at 570 nm, indicated that our scaffold could well barricade P. aeruginosa bio lm formation on its surface. This is visible in the 96 micro-well plate. The reported values are the average of three independent repetitions of the experiment.

4-Conclusions
In this study, Graphene oxide-lignin/silk broin/ZnO nanobiocomposite was synthesized for the rst time and the structure was evaluated using FT-IR, EDX, FE-SEM, XRD, and TGA. In addition, the biological characteristics of the synthesized structure were examined. According to the obtained results, this novel nanobiocomposite acted signi cantly as an anti-bio lm agent against P. aeruginosa. Moreover, the MTT assay shows that the viability percentages of Hu02 cells treated with Graphene oxide-lignin/silk broin/ZnO nanobiocomposite after 24, 48, and 72 h of incubation were 89.96%, 89.32%, and 91.28% and the structure is non-toxic. On the other hand, it's compatible with blood because the hemolysis percentage of the synthesized structure after 24 h and 72 h of extraction was 9.5% and 11.76% respectively. Figure 1 Graphical representation of Graphene oxide-lignin/silk broin/ZnO nanobiocomposite synthesis procedure.          Anti-bio lm histogram of polystyrene and Graphene oxide-lignin/silk broin/ZnO nanobiocomposite pieces (** = signi cant, P ≤ 0.05), comes with 96-well plate image.