# The Exploitation of Lysosomes in Cancer Therapy with Graphene-Based Nanomaterials
## 5 The Role of Lysosomes in GNM-Based Cancer Therapy
Moreover, electrostatic repulsion between the protonated DOX and positively charged (charge-reversed in low pH) NGO-PEG-DA also contributed to DOX release. The NGO-PEG-DA/DOX complex exhibited a synergistic anticancer effect of DOX-dependent chemotherapy and NIR-irradiated NGO-dependent photothermal therapy [40]. [PERSON] and colleagues conjugated poly(ethylene imine)-poly(l-lysine)-poly(l-glutamic acid) (PKE) with charge-conversion properties, a proton sponge polymer PEI, and rGO. The resulting PK5E7/PEI-rGO)/DOX complex was negatively charged at pH 7.4 and positively charged at pH 6. The DOX-loaded complex rapidly released the anticancer drug under lysosomal conditions due to electrostatic repulsion between the positively charged DOX and PEI-rGO. PK5E7/PEI-rGO) showed potent anticancer activity in HeLa and A549 lung adenocarcinoma cells under mildly acidic conditions mimicking the tumor microenvironment [39]. In the presence of the anticancer drug cisplatin, [PERSON] et al. transformed 2D sheets of GO, bound to the DNA-damaging drugs proflavin or DOX by \(\pi\)-\(\pi\) interaction, into 3D spherical GPC-NP and GDC-NP complexes, respectively. After clathrin-mediated endocytosis in HeLa cells, proflavin and DOX were released from the complexes under the influence of lysosomal acidity. Carboxylate bonds were broken, and cisplatin was also released from the complexes at low pH, returning the 3D GO to its 2D form. Ultimately, the cells died by apoptosis [61]. [PERSON] et al. developed a mitochondria-targeting carrier (GT) by attaching the amphiphilic polymer DSPE-PEG (composed of 1,2-distearoyl-3-phosphatidylylethanolamine (DSPE) as the hydrophobic tail and PEG as the hydrophilic chain) to the mitochondria-targeting moiety alkyl triphenylphosphonium (TPP) and GO. The resulting GT was then combined with the immunoconjugate diphenylmethylsilane-substituted CPG (DP-CpG) and the photosensitizer IR820. The formed GT/IR820/DP-CpG complex destabilized lysosomal membrane, escaped lysosomes, and targeted mitochondria of EMT6 mouse breast cancer cells. Upon NIR irradiation, the complex generated heat and ROS, leading to apoptosis. In EMT6 tumor-bearing mice, GT/IR820/DP-CpG produced PDT- and PTT-mediated anticancer effects and activated an anticancer immune response [64]. From previously mentioned studies, we can see that the authors mainly dealt with the separation of antitumor drugs from GNM-containing nanocarriers. The ability of acidic lysosomal milieu to induce physiochemical changes in nanocomplexes was mainly used for that purpose. With the exception of positively charged ICG/DOX/GO-PPF68 [13], all other complexes were negatively charged but became positive after protonation inside the lysosomes, which induced charge repulsion and separation of the drugs from the rest of the complexes [12; 14; 39; 40; 60]. While the ability of low lysosomal pH to provoke break downs of \(\pi\)-\(\pi\) interactions [59], amide [14], and carboxylate [61] bonds, as well as the ability ROS produced by NIR-irradiated photosensitizer to disrupt diselenide bonds, was exploited, there has been no study that clearly utilized the presence of hydrolytic enzymes for this purpose. Mechanisms of lysosomal drug escape have only been superficially addressed. While the drug escape through the lysosomal membrane, which has been damaged by the heat generated by NIR-irradiated GNMs or the combined effects of heat and ROS produced by NIR-irradiated photosensizers, has been clearly established, [62; 64], the proton sponge mechanism was more hypothesized due to the presence of the ensosomolytic molecule PAMAM than proven [12; 13; 15]. Moreover, lysosomal escape was stated, but its mechanism was not investigated in the rest of the above-mentioned studies [14; 39; 40; 61; 59; 60].