Rther improves its drug loading/delivery capacity [30,31]. GO functionalization with polyethylene glycol (PEG) demonstrates higher delivery efficiency and controllable release of proteins, bio imaging agents, chemotherapeutics, and antiAztreonam site cancer drugs. GO EG features a very good biological safety profile [18,324] and exhibits higher NIR absorbance and capacity in photothermalNanomaterials 2021, 11,3 oftreatment [35]. Recently, we studied the physicochemical traits of PEGylated GO and introduced GO EG in mixture with NIR irradiation as a biocompatible wise nanocarrier in colon cancer cells with enhanced physicochemical properties and greater biological compatibility [36]. In an additional study, we additional expanded these experiments and demonstrated that this modification of GO leads to enhanced biocompatibility of GO EG for human blood cells too [37]. Here, we discuss our recent results on the bioactivity of PEGylated GO NPs in mixture with NIR irradiation on colorectal cancer cells. We performed experiments that aim to reveal the molecular mechanisms of action of this nanocarrier combined with nearinfrared light (NIR) on the high invasive Colon26 along with the low invasive HT29 colon cancer cell lines. In the course of reaching the cancer cells the phototoxicity of GO EG is modulated by NIR laser irradiation. We investigated the cyto-, geno- and mitotoxicity in the cells, treated with GO EG with NIR to prove the biocompatibility in the proposed nanocarrier. We additional studied the prospective of GO EG in mixture with NIR to modulate the activity of certain stress-responsive genes. Our results demonstrate the possible of GO EG bioactivity inside the improvement of nanosystems for colorectal cancer treatments. 2. Materials and Procedures 2.1. Preparation of Poly(Ethylene Glycol)-Modified Graphene Oxide (GO EG) and Physicochemical Characterization of NPs Preparation of GO EG NPs was performed making use of pristine GO (Graphenea, Spain) and mPEG-NH2 (Abbexa Ltd., Cambridge, UK) following a previously established process of [38]. A comprehensive description of the PEGylation of GO and its physicochemical characterization was carried out in our preceding publications [36,39]. Dynamic Light Scattering (DLS, Zetasizer, Malvern Instrument, Ltd., Worcestershire, UK) was utilised to ascertain particles size distributions, typical particle size, zeta prospective and polydispersity index (PDI) of GO and GO EG NPs; transmission electron microscope (TEM, JEM-2100, Tokyo, Japan) was applied to analyze the nanoparticles’ morphology; and UV-Vis spectrophotometer (Specord 210 Plus, Edition 2010, Analytik Jena AG, Thuringia, Germany)–to measure adsorption spectra of both NPs in NIR region. 2.2. Cell Cultures, Media and Treatment Protocols HT29 is really a cell line derived from human colorectal cancer cells (ATCC, HTB-38) even though Colon26 is derived from a mouse colon adenocarcinoma (ATCC, FAUC 365 Description CRL-2638). Each cell lines have been grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with ten (v/v) fetal bovine serum (Sigma-Aldrich, Darmstadt, Germany) and 1 (v/v) a mixture of antibiotics (104 IU penicillin and 104 streptomycin, Sigma-Aldrich, Germany). The cells were incubated at 37 C with 5 CO2 and 95 humidity. Cells were passaged inside the exponentially developing phase every single second day, applying 0.05 trypsin and 0.02 EDTA. Cells had been seeded at a density of two.five 104 cells/well in 96- or 6-well plates and 24 h after seeding the cells had been treated with one hundred /mL GO or GO EG. Cells had been grown under these co.