Liposomal Encapsulation\nof Chlorambucil with a Terpyridine-Based,\nGlutathione-Targeted Optical Probe Facilitates Cell Entry and Cancer\nCell Death

The nitrogen mustard alkylating agent chlorambucil (CBL)\nis a critical\ncomponent of chemotherapeutic regimens used in the treatment of chronic\nlymphocytic leukemia. The cancer cell-killing actions of CBL are limited\nby glutathione (GSH) conjugation, a process catalyzed by the GSH transferase\nhGSTA1-1 that triggers CBL efflux from cells. In the cancer cell microenvironment,\nintracellular GSH levels are elevated to counterbalance oxidative\nstress generated due to the high glycolytic demand. As many chemotherapeutic\ndrugs trigger cell death through mechanisms that depend on reactive\noxygen species (ROS), antioxidant capacity in cancer cells also represents\na barrier to anticancer therapies. Here, we demonstrate that a heightened\nGSH content in cancer cells can also be exploited for cell-selective\ndrug delivery. We successfully synthesized a malononitrile conjugate\nterpyridine-based derivative <b>L1</b>, which specifically reacts\nwith GSH in the presence of other biologically relevant amino acids\nincluding cysteine (Cys) and homocysteine (Hcy). The significant change\nin the electronic spectra of <b>L1</b> in the presence of GSH\nconfirmed GSH detection, which was further corroborated by density\nfunctional theory calculations. We next encapsulated CBL into <b>L1</b>-containing, anthracene-functionalized, and 10,12-pentacosadiynoic\nacid (PCDA)- and 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphocholine\n(DMPC)-based liposomes (<b>Lip-CBL-L1</b>). We established successful\nCBL encapsulation and release from <b>L1</b>-containing liposomes\nin GSH-enriched cancer cells in vitro. Both <b>Lip-CBL-L1</b> and the <b>L1</b>-lacking <b>Lip-CBL</b> control displayed\ncell-killing activity. However, human triple-negative breast cancer\ncells MDAMB231, human lung cancer cells A549, and murine leukemic\nWEHI cells were more sensitive to the cytotoxic effects of <b>Lip-CBL-L1</b> compared to the nonmalignant cells (AC16 and HEK293). Indeed, in\nthese cancer cell lines, <b>Lip-CBL-L1</b> induced greater ROS\ngeneration compared to that of <b>Lip-CBL</b>. Together, our\nresults provide initial evidence of the feasibility of exploiting\nthe unique oxidant environment of cancer cells for optimized drug\ndelivery.