# The Exploitation of Lysosomes in Cancer Therapy with Graphene-Based Nanomaterials
## 4 The Transport of GNMs to Lysosomes  
When solid tumors grow larger than 2 mm in diameter, physical pressure on blood vessels reduces blood flow, resulting in a hypoxic state. To overcome this, tumor cells produce angiogenic factors to form new blood vessels. However, the newly formed blood vessels have an abnormal structure with a lack of smooth muscle layer, while pericytes surrounding the capillaries have large pores. Additionally, lymphatic drainage is limited in large tumors. Nanoscale drugs such as GNMs leak preferentially into tumor tissue through permeable tumor vessels and are then retained in the tumor due to reduced lymphatic drainage. This phenomenon is known as the enhanced permeability and retention (EPR) effect [31; 32]. Next, GNMs come into contact with the surface of cancer cells, either passively or by active targeting; for example, when coupled to ligands for folate, transferrin, or epidermal growth factor receptors overexpressed on tumor cells [16; 33; 34], or by magnetic targeting [35]. The ability and mechanism of entry of nanoparticles into cells depend on several factors, including the type, size, and shape of nanoparticles, as well as the type of cell. Nanoparticles with a diameter between 150 nm and 3 um can more easily pass through the plasma membrane compared to particles less than 150 nm or greater than 3 um in diameter [36]. Cubic particles have been shown to enter the cell more efficiently than cylindrical particles [36], while hydrophilic GO enters the cell more readily than hydrophobic rGO [37]. In addition, positively charged nanoparticles pass through the negatively charged plasma membrane faster than neutral or negatively charged ones [38]. The slightly acidic condition of pH \(\approx\) 6.8 in the tumor microenvironment, which is caused by anaerobic metabolism and accumulation of lactic acid [39], can cause negative GNMs to undergo charge reversal and become positively charged, facilitating their entry into cells [40]. Small hydrophobic graphene sheets and graphene flakes can penetrate the plasma membrane directly [41], while most other GNMs enter the cell by endocytosis. Endocytosis is an energy-dependent process by which cells take up external material by engulfing it in a vesicle derived from the cell membrane. There are different types of endocytosis, including clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and phagocytosis [42]. Small GO nanosheets with a diameter of 0.4 um mainly enter cells via clathrin-mediated endocytosis, which is facilitated by cell surface receptors and results in the formation of clathrin-coated vesicles. On the other hand, large GO nanosheets with a diameter of 0.6-0.8 um are recognized as foreign particles and are internalized through phagocytosis, where they are enclosed in an internal compartment called a phagosome [6]. GQDs cross cell membranes either via clathrin- or caveolae-mediated endocytosis [7]. The internalization of nanoparticles in caveolae-mediated endocytosis begins at the lipid rafts by the formation of caveolae vesicles [43]. Some studies suggest that rGO cannot penetrate the membrane [37], while others suggest that it is taken up by the cell through endocytosis [8] (Figure 2). Nanoparticles that are internalized through caveolae-mediated endocytosis can be directed towards the Golgi, endoplasmic reticulum, or endosomes, forming a caveosome. This compartment usually avoids lysosomal fusion and digestion [44]. Conversely, nanoparticles that enter the cell through clathrin-coated vesicles and phagosomes are typically transported to lysosomes [45]. Clathrin-coated vesicles transport particles through early endosomes (pH 6.5), late endosomes (pH 5.5), and lysosomes (pH 4.5) [45]. Phagosomes fuse with the lysosomes, forming phagolysosomes [46]. Additionally, cytoplasmic GNMs can be captured in autophagosomes and subsequently enter autolysosomes [47; 48] (Figure 2). The degradation of GNMs in lysosomes is still uncertain.