Enhanced Intracellular Delivery of the Reactive Oxygen Species (ROS)-Generating Copper Chelator D-Penicillamine via a Novel Gelatin−D-Penicillamine Conjugate

Abstract

D-Penicillamine (D-pen) is an established copper chelator. We have recently shown that the copper-catalyzed D-pen oxidation generates concentration-dependent hydrogen peroxide (H 2O 2). Additionally, D-pen coincubated with cupric sulfate resulted in cytotoxicity in human leukemia and breast cancer cells due to the extracellular generation of reactive oxygen species (ROS). The inherent physicochemical properties of D-pen such as its short in vivo half-life, low partition coefficient, and rapid metal catalyzed oxidation limit its intracellular uptake and the potential utility as an anticancer agent in vivo. Therefore, to enhance the intracellular delivery and to protect the thiol moiety of D-pen, we designed, synthesized, and evaluated a novel gelatin-D-pen conjugate. D-pen was covalently coupled to gelatin with a biologically reversible disulfide bond with the aid of a heterobifunctional cross-linker ( N-succinimidyl-3-(2-pyridyldithio)-propionate) (SPDP). Additionally, fluorescein-labeled gelatin-D-pen conjugate was synthesized for cell uptake studies. D-pen alone was shown not to enter leukemia cells. In contrast, the qualitative intracellular uptake of the conjugate in human leukemia cells (HL-60) was shown with confocal microscopy. The conjugate exhibited slow cell uptake (over the period of 48 to 72 h). A novel HPLC assay was developed to simultaneously quantify both D-pen and glutathione in a single run. The conjugate was shown to completely release D-pen in the presence of glutathione (1 mM) in approximately 3 h in PBS buffer, pH 7.4. The gelatin-D-pen conjugate resulted in significantly greater cytotoxicity compared to free D-pen, gelatin alone, and a physical mixture of gelatin and D-pen in human leukemia cells. Further studies are warranted to assess the potential of D-pen conjugate in the delivery of D-pen as a ROS generating anticancer agent.

Copper as the Target for Anticancer Nanomedicine

“…[127] To improve the intracellular delivery and to protect the thiol moiety of D-pen, Mumper et al conjugated D-pen with gelatin (gelatin-D-pen) and poly-L-glutamic acid (PGA-D-pen) both via reversible disulfide bonds. [127] To improve the intracellular delivery and to protect the thiol moiety of D-pen, Mumper et al conjugated D-pen with gelatin (gelatin-D-pen) and poly-L-glutamic acid (PGA-D-pen) both via reversible disulfide bonds.…”

“…[127] To improve the intracellular delivery and to protect the thiol moiety of D-pen, Mumper et al conjugated D-pen with gelatin (gelatin-D-pen) and poly-L-glutamic acid (PGA-D-pen) both via reversible disulfide bonds. [127,128] Based on the high binding affinity between sulfur and copper ions, Zheng et al developed sulfur nanoparticles (Nano-S) as Cu chelators to inhibit the growth of cancer cells. [127,128] Based on the high binding affinity between sulfur and copper ions, Zheng et al developed sulfur nanoparticles (Nano-S) as Cu chelators to inhibit the growth of cancer cells.…”

“…Due to its high hydrophilicity and its reactive thiol group, D-pen is prone to oxidation to D-pen disulfide in vivo and rapid removal by kidneys. [127] To improve the intracellular delivery and to protect the thiol moiety of D-pen, Mumper et al conjugated D-pen with gelatin (gelatin-D-pen) and poly-L-glutamic acid (PGA-D-pen) both via reversible disulfide bonds. [127][128][129] Although both could achieve the glutathione (GSH)-responsive release of D-pen, PGA-D-pen was cytotoxic to cancer cells and significantly enhanced the survival of mice, whereas gelatin-D-pen showed low anticancer efficacy, which might be caused by the difference in the cellular uptake rate.…”

“…[127] To improve the intracellular delivery and to protect the thiol moiety of D-pen, Mumper et al conjugated D-pen with gelatin (gelatin-D-pen) and poly-L-glutamic acid (PGA-D-pen) both via reversible disulfide bonds. [127][128][129] Although both could achieve the glutathione (GSH)-responsive release of D-pen, PGA-D-pen was cytotoxic to cancer cells and significantly enhanced the survival of mice, whereas gelatin-D-pen showed low anticancer efficacy, which might be caused by the difference in the cellular uptake rate. [127,128] Based on the high binding affinity between sulfur and copper ions, Zheng et al developed sulfur nanoparticles (Nano-S) as Cu chelators to inhibit the growth of cancer cells.…”

“…[127][128][129] Although both could achieve the glutathione (GSH)-responsive release of D-pen, PGA-D-pen was cytotoxic to cancer cells and significantly enhanced the survival of mice, whereas gelatin-D-pen showed low anticancer efficacy, which might be caused by the difference in the cellular uptake rate. [127,128] Based on the high binding affinity between sulfur and copper ions, Zheng et al developed sulfur nanoparticles (Nano-S) as Cu chelators to inhibit the growth of cancer cells. The chelation of copper by Nano-S resulted in a tight surficial structure, which inhibited the activation of the MEK/ERK signaling and the growth of A375 melanoma cells and MCF-7 breast cancer cells.…”