# The Exploitation of Lysosomes in Cancer Therapy with Graphene-Based Nanomaterials
## 5 The Role of Lysosomes in GNM-Based Cancer Therapy
Although the fact that antitumor drugs escaped from lysosomes seems to be more important for their antitumor activities than how they escaped from lysosomes, an elucidation of the mechanisms of escape would be a useful guide for future synthesis of graphene-based DDSs and should, therefore, be clearly elucidated.5.2. GNMs Induce Lysosomal Cell Death Lysosomal escape may be associated with LMP. The consequence of LMP is the leakage of lysosomal enzymes into the cytoplasm. Cathepsins B, D, and L retain their activity at neutral pH, degrading cytoplasmic proteins and inducing LCD. The type of LCD induced depends on the severity of lysosomal damage; extensive LMP, with leakage of cathepsin and protons into the cytoplasm, triggers necrosis, while limited LMP triggers apoptosis. Additionally, LMP may also play a role in the initiation of autophagic cell death, necroptosis, and ferroptosis [3]. LCD may occur subsequent to the escape of nanoparticles from lysosomes via various mechanisms, including the proton sponge effect, the umbrella effect, pore formation, or photochemical disruption of the lysosomal membrane [56; 66; 67]. However, in some cases, LCD may not occur due to the dominance of other mechanisms of cell death. For instance, a study involving the treatment of tumor cells with ICG/DOX/GO-PPF68 detected clear LMP, but the authors attributed the antitumor effects to the antiproliferative activity of DOX, ROS generated by NIR-irradiated ICG, and hyperthermia caused by NIR-irradiated GO and ICG [15]. Furthermore, in another study, GO nanosheet-induced LMP led to lysosomalalkalinization, which inhibited autophagic degradation and, ultimately, triggered apoptosis, but not LCD [10]. It is crucial to distinguish between endosomal and lysosomal escape as endosomes have a lower acidity level and hydrolytic enzymes content. Consequently, LCD is more likely if the drug escapes from mature lysosomes rather than endosomes [56]. It is important to note that escape of anticancer drugs through pore formation may not necessarily result in the leakage of cathepsin. The formed pores may be large enough to allow for the release of the drug but not the hydrolytic enzymes. For example, Cathepsin B has a molecular weight of approximately 25,000 Da [68], while the molecular weight of DOX is only 544 Da [69]. Oxidative stress is one of the main LMP inducers [3]. Accelerated metabolism of tumor cells results in increased breakdown of iron-containing proteins and iron accumulation in lysosomes. This iron can convert ROS into highly reactive OH\(\bullet\) via the Fenton reaction [70], which in turn disrupts lysosomal membrane integrity. Despite its short half-life, OH\(\bullet\) is a very toxic molecule due to its high reactivity. OH\(\bullet\) causes breaks in DNA strands, modifies DNA bases, and disrupts protein structure and function. OH\(\bullet\) also triggers the peroxidation of lipids, which are essential components of cell membranes, including lysosomal membranes [71]. Lysosomes lack the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase [3], making them particularly vulnerable to oxidative stress. Accordingly, oxidative stress has been identified as the mediator of LMP in all studies involving both GNMs and LMP (Table 2). In a study conducted by [PERSON] et al., rat basophilic leukemia RBL2H3 cells endocytosed graphene nanosheets (GNSs), which, due to their sharp edges and rough surface, damaged the lysosomal membrane, thereby inducing LMP. GNSs also disrupted the mitochondrial electron transport chain, leading to excessive ROS production. This oxidative stress further stimulated LMP and mitochondrial membrane depolarization, ultimately resulting in apoptotic cell death [67]. [PERSON] et al. developed a complex by assembling 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (DSPE-PEG2000-FA) and a photosensitizer-labeled peptide (Ce6-Pep) on the surface of GO. This complex was internalized by HeLa cells via folate receptor-mediated endocytosis and localized in lysosomes.