## 3 Results and Discussion
### Biological Assays
#### 3.5.2 Cellular Uptake and Intracellular Localisation  
The cell membrane employs different mechanisms to exchange substances, including passive and active transport as described previously. Endocytosis is a type of active transport and is where the cell engulfs external objects inside the extracellular fluid. Endocytosis is employed when NPs cannot pass through by passive transport due to either the size, charge, or polarity of the nanoparticle [95; 96]. Twelve of the publications evaluated the cellular uptake of DOX into ovarian or breast cancer cell lines by means of fluorescent visualisation. Of these studies, 75% suggested that the mechanism of cellular uptake was endocytosis. The fact that DOX is intrinsically fluorescent negates the need for additional fluorescent probes. This is beneficial, as introducing additional elements to the nanocarrier increases the complexity and decreases the clinical translatability [71].  
The mechanism of endocytosis employed in each of the studies will be dependent on the size, shape, surface charge, surface hydrophobicity, and functionalisation of nanocarriers. Folic acid is utilised in several publications for enhanced targeting, as it facilitates endocytosis through FR-mediated endocytosis. [PERSON] et al. evaluated the uptake of DOX in MCF-7 cells with high levels of FR and human non-small-cell lung cancer cell line A549, which lack FR. The difference in uptake of FA-BSA/GO/DOX compared to the non-specific BSA/GO/DOX was significant in the MCF-7 cells, but was similar when incubated with A549 cells; thereby suggesting the folate-mediated endocytosis [70]. Alternatively, [PERSON] et al. demonstrated specific cellular uptake of the nanohybrid GO-HA-RGD through both antigen and integrin binding. HA can specifically bind to the overexpressed antigen CD44 on the surface of tumour cells. In addition, the RGD targeting peptide recognises specific integrins on the tumour cell surface. The synergic effect of CD44-HA and Integrin-RGD mediated endocytosis facilitates the successful uptake into SKOV-3 cells [44].  
By utilising receptor-mediated endocytosis and antibody-mediated endocytosis, the specificity of cellular targeting and subsequent uptake is improved. In living systems, off-target cells that are diseased or damaged may also have an acidic pH, be hypoxic, or be inflamed like the target cancer cells. Receptor- or antibody-mediated endocytosis reduces the chance of cellular uptake by non-specific target cells.  
Sub-cellular targeting and intracellular localisation of graphene and its derivates have not been extensively explored. It is important to understand where the DOX-loaded graphene nanocarrier accumulates within the cell and to identify if any functional groups elicit any specific targeting of intracellular organelles. Most of the publications suggested that the DOX accumulates in both the nucleus and cytoplasm, although the localisation was not comprehensively studied throughout. There is most likely a high accumulation of DOX in the nuclei of cells due to the high affinity it has for DNA [77]. In addition, [PERSON] et al. functionalised a graphene nanocarrier with hypericin, a mitochondria-targeting ligand, and found it to elicit an anti-tumour effect by inducing the mitochondria-mediated apoptosis pathway [66].