Methods

0.5 g of GG powder was dispersed in 50 mL distilled water in a 200 mL beaker at room temperature. Separately, 0.5 g AG powder was dispersed in 50 mL distilled water and added to the GG solution. The mixture was stirred continuously at 70–80 °C for 30 min and then, ZnCl₂ was dissolved in 20 mL of distilled water, sonicated for 30 s and added to the above mixture. After 12 h, the produced hydrogel was collected, cooled at room temperature and freeze dried for analyses.

g-C₃N₄ was prepared based on previously reported methods⁴⁰. Firstly, urea was dried for 24 h at 80 °C oven and was heated at 550 °C oven for 3 h in a covered crucible. The yellow powder of g-C₃N₄ was collected after the mentioned time. 5 mL of the prepared GG–AG hydrogel was mixed with 0.02 g g-C₃N₄ powder and was stirred continuously at room temperature for 30 min. 5 mL of the prepared composite was collected for the next step and the rest of the composite was collected and freeze dried for analyses.

1 g Zn(NO₃)₂·6H₂O, 1.3 g Fe(NO₃)₃·9H₂O and 1 g Cu(NO₃)₂ were mixed and added to 100 mL distilled water and sonicated for 30 s. Afterwards the mixture was stirred at room temperature for 20 min. 0.5 g NaOH was dissolved in 10 mL distilled water and added to the above mixture. The solution was stirred at 50 °C for 3 h and after the mentioned time, the mixture was kept in 150 °C oven for 4 h in a stainless-steel autoclave. Afterwards, it was washed with distilled water and ethanol for several times, centrifuged and dried at 60 °C oven overnight. 0.02 g of the prepared ZnCuFe₂O₄ was added to 5 mL GG–AG hydrogel/g-C₃N₄ composite and stirred at room temperature for 30 min. The final composite was collected and freeze dried for further investigations.

TG analysis was performed using Bahr-STA 504 instrument (Germany). In order to perform the TGA, 5.0 mg of the sample was transferred into alumina pans under argon atmosphere and 1 L/h flow rate. Heating range was between 50 and 600 °C with 10 °C/min rate.

The elemental composition of the nanobiocomposite was determined by EDX analysis (SAMx model, France) with ultrathin window detector.

For morphological identification of the samples, they were subjected to FE-SEM analysis (ZEISS-Sigma VP model, Germany) at 15 kV. Samples were mounted on stainless-steel stub by double side carbon tape (Agar Sputter Coater model, Agar scientific, England).

FT-IR analysis was performed by (Shimadzu FT-8400s model, Japan), by using KBr pellet preparation method. 0.1–1.0% of each sample was mixed with 200–250 mg KBr and the prepared pellets were subjected to FT-IR frequency range of 400–4000 cm⁻¹ at 25 °C.

XRD was performed using PANalytical X-PERT-PRO MPD at 2Ɵ = 5°–85° with STA504 analyzer in the temperature range of 50°–550° and in air (10 °C/min).

The synthesized GO/Casein/LDH/Alg/Fe₃O₄ nanobiocomposite was extracted by dispersing 50 mg of it in 1 ml of phosphate buffer saline (PBS) using shaker incubator for 48 h at 37 °C.

MTT assay was performed to determine the biocompatibility of nanobiocomposite. For this purpose, HEK293T cells were firstly cultured in DMEM/F12 medium with 10% FBS. Afterwards, 5 × 10³ cells/well were moved to 96-well plates and 10 μL of the nanobiocomposite extract was transferred into each cell and incubated for 24 h, 48 h and 72 h. PBS-treated cells were also considered as negative control. Afterwards, cells were treated with MTT (3-4,5dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) (Sigma, USA) and incubated for another 4h at 37 °C. 1% SDS was added to cell/wells and incubated at 37 °C for 16h. By using a microplate reader spectrometer (BioTek. USA) at 550 nm, optical densities (OD) were measured and cell viability was assessed using following formulas⁴¹:

Antibacterial activity of the prepared nanobiocomposite was studied using tissue culture plate (TCP) anti-biofilm assay. For this purpose, 1 cm² of the nanobiocomposite was sterilized in 70% ethanol alongside with a polystyrene piece as the positive control, and were dried at 37 °C in an incubator. Each piece was then transferred to sterilized tubes containing Pseudomonas aeruginosa bacteria (ATCC 27853)with 10⁷ colony-forming unit (CFU)/mL concentration in a culture medium of Nutrient Brot (NB). Afterwards, tubes were incubated for 24 h at 37 °C in a shaker incubator with shake speed 150 rpm. Samples were washed with PBS and stained by 0.1% crystal violet solution for 5 min and then were washed with 33% acetic acid solution. Finally, by using a microplate reader (STAT FAX 2100, BioTek, Winooski, USA), the resulting solution’s OD was evaluated at 570 nm⁴².

Statistical analysis for the comparison all results was accomplished by a t-test by SPSS Statistics 22.0 software (SPSS Inc. Chicago, IL, USA). The values of P ≥ 0.05 (*), P ≤ 0.05 (**) and P ≤ 0.001 (***) were considered as statistically insignificant, significant and very significant, respectively.

The samples (hydrogel and final nanobiocomposite) were swollen in ultra-pure water at room temperature for 24 h before the rheological measurements were taken using an RMS/MCR 302 rheometer (Anton-Paar Co., USA) equipped with a 20 mm parallel plate. Measurement of the storage modulus (G′) and loss modulus (G″) were conducted at shear stress range from 0.01 to 1000 Pa at controlled frequency of 0.1 Hz. The measurements were stopped when both G′ and G″ began to decrease notably. For this test, three specimens were measured, and the values were averaged. Dynamical mechanical analysis of the swollen samples was performed by changing the oscillatory stress from 0.01 to 1000 Pa at a constant frequency (1 Hz)⁴³.

The compressive mechanical properties of GG–AG hydrogel/g-C₃N₄/ZnCuFe₂O₄ nanobiocomposite, were measured according to the procedure of Bhardwaj et al. and ASTM method F451-95 with some modifications⁴³. This test was performed using a Universal Testing machine (SANTAM, STM-20 model) with a load cell capacity of 0.1 kN with crosshead speed 1 mm/min at room temperature. Based on this method, the specimens with a thickness of 10 mm and a diameter of 13 mm were cut manually from nanobiocomposite using a razor blade. In the following, the samples were soaked in PBS solution for 2 h and this test was accomplished under wet conditions. At least three fragments were tested and the mean values were reported.

Freeze-dried nanobiocomposite was immersed in UPW at 25 °C for 48 h. Surplus UPW was then removed from the surface of sample and the wet weight of the nanobiocomposite was determined. The swelling ratio and the water uptake in the sample were calculated as follows:

In this formula, W_s and W_d are the weights of dried and swollen nanobiocomposite, respectively⁴⁴.

For degradation experiments, GG–AG hydrogel/g-C₃N₄/ZnCuFe₂O₄ nanobiocomposite was placed into PBS at pH = 7.4 and 37  °C The buffer solution was refreshed every 3 days. This test was performed up to 10 days and at the selected time points, three samples of nanobiocomposite were removed from the buffer and weighed wet after surface wiping. Afterwards, they were rinsed with UPW and dried in a vacuum oven at 37 °C for 24 h. Water absorption and weight loss were calculated according to these formulas:

where W0 is the starting dry weight, Wa is the wet sample weight after removal from the solution, and Wt is the dry sample weight after removal⁴⁵.