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Pharmaceutics 2023, 15, 993 3 of 15 Inc. (Waltham, MA, USA). Precision Plus Protein Dual Color Marker and Clarity Western ECL Substrate were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Pri- mary rabbit monoclonal antibodies to HO-1 (1:750) to NF-κB (1:1000) and NRF2 (1:750), as well as secondary rabbit-specific horseradish peroxidase-conjugated antibody (1:1000), were all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary mouse monoclonal antibody to β- tubulin (1:500) and secondary mouse-specific horseradish peroxidase-conjugated antibodies (1:1000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2.2. Synthesis of Bio-Graphene (bG) For the synthesis of bio-graphene (bG), a suspension of 100 mg of graphite in 20 mL of double distilled water was subjected to ultra-sonication for one hour (200 W, 10 kHz and pulser 50%). Next, 100 mg BSA (dissolved in 5 mL of double distilled water) was transferred to the partially exfoliated graphitic sheets, and the graphite–BSA solution was incubated at room temperature under stirring for 1 h. These steps result in the production of few-layered bG, where BSA is used as both the exfoliation agent and the stabilizer of the graphitic sheets, as recently published [23]. Herein, the next step was slightly modified, involving the centrifugation of the mixture at 2500 rpm for 10 min to separate the non-exfoliated graphite. The supernatant was carefully separated from the non-exfoliated graphite sheets. The supernatant acquired after the centrifugation was freeze-dried, and the powder was gathered and weighed. The concentration of bG was calculated using the equation: Concentration (mg/mL) = mg of freeze-dried powder/mL of supernatant 2.3. Synthesis of Chemical-Graphene (cG) The chemical production of graphene sheets entails the utilization of graphite as a starting material. There are various methods for the chemical exfoliation of graphite into graphene [24]. The oxidation of graphite [25,26] happens via the help of concentrated acids (such as sulfuric, nitric, etc.) and a strong oxidizing agent (e.g., potassium chlorate or potassium permanganate [26]) leading to high-quality GO by the simple mixing of acids. The resulting GO possesses a large variety of oxygenated functional groups such as carboxyl, epoxide, hydroxyl, etc. depending on the oxidation method followed while the synthesis does not require thermal treatment. GO is transformed into graphene through a reduction treatment [27] to obtain high-quality graphene. The reduction takes place by the dispersion of GO in water and the subsequent addition of NaHB4 while the mixture is stirred in a steam bath for 3 h. The second path describes the delamination of graphite into graphene without the need for any oxidation or functionalization method by liquid phase exfoliation. This method lies in the fact that the solvent–graphene interaction is the same or similar to the interactions of stacked graphene layers in graphite. Among a high number of suitable solvents that can exfoliate graphite (N-methylpyrrolidone, N, N-dimethylacetamide, g-butyrolactone and perfluorinated aromatic molecules), DMF is one of the most common solvents used for this method with very good performance [28]. 2.4. Cell Lines An immortal keratinocyte cell line derived from adult human skin (HaCaT cell line, CLS GmbH, 300493), a fibroblast cell line isolated from a mouse NIH/Swiss embryo (NIH/3 T3 cell line, ATCC, CRL-1658) and a human monocyte cell line from a patient with acute monocytic leukemia (THP-1 cell line, DSMZ, ACC16) were used in this study. HaCaT and NIH/3T3 cells were cultured with high glucose Dulbecco’s modified eagle medium and THP-1 with RPMI-1640 medium. Both mediums were supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) L-glutamine and 1% (v/v) penicillin–streptomycin solution. All cell lines were grown in a humidified incubator (5% CO2, 95% air) at 37 ◦C. Prior to all experiments, the THP-1 monocytes were differentiated into mature macrophages with phorbol 12-myristate 13-acetate (PMA) at a concentration of 100 ng/mL for 24 h [29].PDF Image | Green Exfoliation of Graphene
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