Here the study demonstrated graphene oxide antimicrobial efficacy against biofilm and intracellular S. aureus isolated from subclinical cases mastitis cases in Malaysia. GO was effective against the extracellular, intracellular, and biofilms forms of S. aureus. The intracellular antimicrobial activities appeared to be dependent on the actin polymerization of the membrane cell that controls the macropinocytosis uptake pathway of the cells. Finally, the study showed that growth of Mac-T cells was enhanced when exposed to low concentrations of GO, and toxicity was only observed when the cell was exposedto very high concentrationsn of GO.
GO antimicrobial activities could be attributed to several mechanism [26]. GO consist high amount of oxygen functional groups that can wrap the bacteria and, at the same restricting nutrient intake inside the cells [36]. The Van der Walls interaction between the functional groups and the membrane can also trigger oxidative stress, destabilize the bacterial membrane, leading to the release of intracellular contents [37]. In addition, the sharp edges of GO can pierce and thus causing injuries to the membrane structure [38].
GO antibiofilm activities were observed, where reduction of biofilm mass was recorded when GO was exposed to biofilms. This data is consistent with the study conducted by Guilio et al. on the GO effect towards biofilms of S. aureus, Pseudomonas aeruginosa, and Candida albicans, the most common organisms associated with chronic wounds [39]. Our current study found that GO at 100 μg/mL has better biofilm activity. Whereas at higher concentrations has lower, this could be due to aggregation of GO that could affect their activity. Physiologically, biofilm structure is built of individual cells that are glued together with extra polymeric substances (EPS), which consist of self-released glycolipids, proteins, lipopolysaccharides, and extracellular DNA. The anti-biofilm effect demonstrated by GO could be attributed to the interaction between the hydrogen, Van der Walls, electrostatic, and pi-pi/ π-π interactions. These characteristics allow for interaction with other molecules, including DNA, proteins, and polymers, and destabilize the EPS structure. Also, the sharp edges of GO sheets could penetrate and tear the EPS structure and thus destabilize the microbial biofilm even further.
The treatment for infections by intracellular S. aureus remains a challenge because antimicrobials need to penetrate the host cells in order to reach the bacteria. S. aureus was reported to zlocalize inside the cell cytosol following the invasion of the host cell [40]. Thus, antimicrobials must not only be able to cross the cell barriers, but it also needs to reach the cytosol where the bacteria reside. Moreover, the mammalian cell membrane is built of the lipid bilayer that allows for selective permeability across its structure [41]. Depending on the physiochemical properties, a compound can either transverse the membrane either through several uptake mechanism. Nanoparticles such as graphene derivatives was reported to be taken up by mammalian cells by several route, depending on the size of the compounds, surface charge as well as the type of cells [41, 42]. For example, graphene quantum dots were reported to be taken up by the breast cancer cells via caveolae mediated endocytosis route [43]. Chen et al. demonstrated that the mechanism for GO uptake into epithelial lung cells, human epithelial skin cell line, human embryonic kidney cell line, and mouse fibroblast cells were size dependent. GO within the size range of 477 nm was taken up by the cells via micropinocytosis, while the smaller sized of GO within 123 nm were mainly taken up by the cells by clathrin and caveolae-mediated endocytosis [44].
In the current study, the uptake mechanism of GO in the bovine mammary cells were indirectly measured by blocking the possible uptake pathway with inhibitors prior to GO exposure toward the infected cell. We anticipated that if GO intracellular antimicrobial were inhibited when cells were pretreated with any of the tested inhibitors, that would suggest the potential route of uptake for the compound into the bovine mammary cells. In this study, reduction in GO efficacy to kill intracellular S. aureus were observed when Mac-T cells were pre-treated by cytochalasin D, a macropinocytosis inhibitor, suggesting that the GO uptake into the bovine mammary Mac-T cells were through macropinocytosis route. Macropinocytosis is a clathrin-independent endocytic uptake mechanism for the non-selective solute, including molecules, nutrients, and antigens, that takes place in both phagocytic and non-phagocytic cells [45]. The process involves actin cytoskeleton rearrangement on the plasma membrane triggered by molecules such as GO. The rearrangement can form membrane ruffles that fold back onto themselves, hence trapping the molecule to be transported inside the cells. The invaginated membrane ruffles containing the molecules, e.g., GO then pinched inside the cells within the vesicular membrane. The pinched-off vesicle can become acidified, and this process can trigger the release of GO into the cytosol [45, 46].
Alternatively, there is a possibility for the vesicular membrane to be pierced by GO’s sharpness structure, promoting its release into the cytosol. Another study, however, suggested a different fate for GO. Lammel et al. demonstrated that once GO was taken up by the hepatocellular carcinoma (HEP G2) cells, it was released directly into the cytosol without being trapped in the vesicular compartment [47]. The same observation was also reported when GO was exposed to fish cell lines [48]. Nonetheless, our data on GO’s efficacy against intracellular S. aureus suggest that the nanomaterial is able to reach the bacteria inside the host cells to exert antimicrobial activities.
In addition, the GO intracellular antimicrobial activities were highly influenced by the types of solvent or solution used during the experiment, where antimicrobial efficacy was only achieved in saline solution but not in DMEM and PBS. The same observation was reported by Hui et al. that showed GO bactericidal activities against E. coli and Bacillus subtilis were only recorded in saline but not in Luria Bertani broth, the common medium used for bacteria culture [49]. Another study by Chen et al. showed GO prepared in PBS showed less antimicrobial efficacy compared to GO prepared in NaCl and water when it was tested against Xanthomonas oryzae pv. Oryzae [50]. Hui et al. suggested that these phenomena could be due to the absorption of molecules in the medium onto the GO surface, which could have masked its edges and functional groups are responsible for the antimicrobial activities.
Additionally, divalent cations such as Ca2+ and Mg2+, which is commonly found in the cell culture medium, could cause aggregation in GO molecules, potentially due to cross bridging between the cations and the functional group and edges of the GO sheet [51]. The aggregation could reduce the surface area for the interaction of GO and the bacteria, limiting its antimicrobial efficacy [52]. This important observation, therefore, highlighted the importance of choosing the right solution for the measurement of GO antimicrobial activities.
In this study, GO unexpectedly enhanced bovine mammary cells at low concentrations, and this phenomenon highlighted the versatility of the nanomaterial. The impact of graphene nanocomposites, particularly GO, on cell growth has been recorded before. GO promoted cell growth and induced differentiation of the different types of stem cells, including bone progenitor cells and mouse embryogenic stem cells. Ruiz et al. demonstrated macrophages grown on the surface of low concentration of GO displayed high attachment and promoted cell growth [53]. The increased surface area with functional groups allows for biomolecules and stem cell attachment on the surface. GO has also been shown to support cell growth by allowing spontaneous differentiation and promoting selective differentiation of progenitor cells [54]. Therefore, this observation showed that GO not only effective to kill intracellular S. aureus, but it also has the potential to aid bovine mammary cell growth.
This study also demonstrated that bovine mammary cells tolerated GO at concentration that exceeded the bactericidal concentration against the bacteria. This data showed that GO is effective as an antimicrobial at high therapeutic index. Several reports were recorded on graphene toxicity different types of mammalian cells. Nguyen et al. exposed a range of GO concentrations (10 to 200 μg/mL) toward human carcinoma epithelial cell lines. At 200 μg/mL, no obvious toxicity of GO were reported [55]. Several other studies measured GO toxicity towards the following cell line, human breast cancer, ovarian cancer, HeLa and mouse embryonic fibroblast. Briefly, GO toxicity level varied and highly dependent on time of exposure and dose of the compound [55,56,57,58,59,60]. Nevertheless, this study showed that Mac-T cells appeared to have tolerance to GO with cell viability were only affected when cells were exposed to GO at concentration higher than 1000 μg/mL, which is higher than the concentration needed to kill intracellular S. aureus in the host cells.
In conclusion, this study demonstrates that GO has antimicrobial activity against biofilm and intracellular S. aureus. GO intracellular antimicrobial activities appeared to be dependent on the actin polymerisation of the Mac-T cells that promotes uptake of the nanomaterial through macropinocytosis route. Finally, GO at low concentration promoted bovine mammary cell growth, and toxicity was only profound when cells were exposed at high concentrations. These findings highlighted GO efficacy and suggested the suitability of the nanomaterial to be further tested and developed as an effective therapy for bovine mastitis.