Liposomal Encapsulation of Chlorambucil with a Terpyridine-Based, Glutathione-Targeted Optical Probe Facilitates Cell Entry and Cancer Cell Death.

Abstract

The nitrogen mustard alkylating agent chlorambucil (CBL) is a critical component of chemotherapeutic regimens used in the treatment of chronic lymphocytic leukemia. The cancer cell-killing actions of CBL are limited by glutathione (GSH) conjugation, a process catalyzed by the GSH transferase hGSTA1-1 that triggers CBL efflux from cells. In the cancer cell microenvironment, intracellular GSH levels are elevated to counterbalance oxidative stress generated due to the high glycolytic demand. As many chemotherapeutic drugs trigger cell death through mechanisms that depend on reactive oxygen species (ROS), antioxidant capacity in cancer cells also represents a barrier to anticancer therapies. Here, we demonstrate that a heightened GSH content in cancer cells can also be exploited for cell-selective drug delivery. We successfully synthesized a malononitrile conjugate terpyridine-based derivative L1, which specifically reacts with GSH in the presence of other biologically relevant amino acids including cysteine (Cys) and homocysteine (Hcy). The significant change in the electronic spectra of L1 in the presence of GSH confirmed GSH detection, which was further corroborated by density functional theory calculations. We next encapsulated CBL into L1-containing, anthracene-functionalized, and 10,12-pentacosadiynoic acid (PCDA)- and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-based liposomes (Lip-CBL-L1). We established successful CBL encapsulation and release from L1-containing liposomes in GSH-enriched cancer cells in vitro. Both Lip-CBL-L1 and the L1-lacking Lip-CBL control displayed cell-killing activity. However, human triple-negative breast cancer cells MDAMB231, human lung cancer cells A549, and murine leukemic WEHI cells were more sensitive to the cytotoxic effects of Lip-CBL-L1 compared to the nonmalignant cells (AC16 and HEK293). Indeed, in these cancer cell lines, Lip-CBL-L1 induced greater ROS generation compared to that of Lip-CBL. Together, our results provide initial evidence of the feasibility of exploiting the unique oxidant environment of cancer cells for optimized drug delivery.