Intracellular Delivery of the Reactive Oxygen Species Generating Agent d-Penicillamine upon Conjugation to Poly-l-glutamic Acid

Abstract

D-penicillamine is an aminothiol that is cytotoxic to cancer cells and generates dose dependent reactive oxygen species (ROS) via copper catalyzed oxidation. However, the delivery of D-pen to cancer cells remains a challenge due to its high hydrophilicity, highly reactive thiol group and impermeability to the cell membrane. To overcome this challenge, we investigated a novel poly-L-glutamic acid (PGA) conjugate of D-pen (PGA-D-pen) where D-pen was conjugated to PGA modified with 2-(2-pyridyldithio)-ethylamine (PDE) via disulfide bonds. Confocal microscopy and cell uptake studies showed that the fluorescently labeled PGA-D-pen was taken up by human leukemia cells (HL-60) in a time dependent manner. Treatment of HL-60, murine leukemia cells (P388) and human breast cancer cells (MDA-MB-468) with PGA-D-pen resulted in dose dependent cytotoxicity and elevation of intracellular ROS levels. PGA-D-pen induced apoptosis in HL-60 cells which was verified by Annexin V binding. The in vivo evaluation of the conjugate in the P388 murine leukemia model (intraperitoneal) resulted in significant enhancement in the survival of CD2F1 mice over vehicle control.

Polymers for medical and tissue engineering applications

“…Poly(L‐glutamic acid) (L‐PGA) is a flexible synthetic poly(amino acid) that is relatively easy to fabricate into different architectures . It is also non‐immunogenic and enzymatically degradable . It has featured as cancer drug delivery and MRI contrast agents and as a copolymer with other polymers in tissue engineering scaffolds.…”

d-Penicillamine targets metastatic melanoma cells with induction of the unfolded protein response (UPR) and Noxa (PMAIP1)-dependent mitochondrial apoptosis

“…Moreover, in a previous phase II clinical trial, DP administered at FDA-approved doses did not improve survival in patients with glioblastoma multiforme [54]. However, more recent drug development efforts have aimed at improving DP chemotherapeutic efficacy by optimization of intracellular delivery, and systemic administration of DP poly-L-glutamic acid conjugates displayed significant activity in a murine leukemia model [55]. Therefore, feasibility of harnessing DP-induced UPR-induction and apoptogenicity for future chemotherapeutic intervention targeting metastatic melanoma remains to be established.…”

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. [127,128] 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.…”

“…[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.…”