(M1530-11-74) Micellar Codelivery of DQ, a Disulfiram Based Prodrug with In Situ Copper Ions Chelation for Cancer Therapy

Purpose: Disulfiram (DSF), an FDA-approved alcoholism medicine, has made the front page of multiple clinical trials for cancer treatment due to drug repurposing. DSF has in the presence of copper ions. One of DSF's metabolites, diethyldithiocarbamate (DTC), chelates copper ions to form the DTC-copper complex (CuET). CuET has strong anticancer activity. However, tumor tissues lack sufficient copper ions to chelate with DTC. Delivery copper ions to targeted tumor tissues could be one solution. Despite the excellent anticancer effect of CuET, DTC and copper codelivery cannot be considered the best solution. This is especially true in normal tissue, where CuET is extremely toxic and there is a lack of selectivity between tumor and normal tissues. DQ is an H₂O₂-responsive DSF prodrug that can release DTC and quinone (QM) in the tumor microenvironment when exposed to H₂O₂ [1]. Given that, a poly(ethylene glycol) methyl ether-branched polyethyleneimine-cholic acid (PEG-PEI-CA) based delivery system was developed for codelivery of DSF based prodrug DQ and sufficient copper ions. Our delivery carrier was rationally designed with poly(ethylene glycol) methyl ether (PEG), a shealth surface material which exerts long circulation in blood. Cholic acid was used as hydrophobic block to encapsulate DQ. Branched polyethyleneimine (BPEI) was repurposed as a copper ion carrier in this study. Given this, a poly(ethylene glycol) methyl ether-branched polyethyleneimine-cholic acid (PEG-PEI-CA)-based delivery system for DSF-based prodrug DQ and adequate copper ions was developed.

Methods: DQ was synthesized based on a previous report [1]. DQ-Cu nanoparticles were encapsulated into PEG-PEI-CA nanoparticles through a thin film dispersion method. Sizes and serum stability of DQ-Cu nanoparticles were then characterized by dynamic light scanning. Calcein fluorescence competition assay was performed to investigate whether DSF could release DQ under H₂O₂
condition or not. Next, in vitro study of different formulations were performed. The cell viabilities of DQ-Cu nanoparticles in different cell lines were evaluated by Gen5 Cytation plate reader. Mitochondrial reactive oxygen species (ROS) level in 4T1 cells after treated with different formulations were measured. The mitochondria membrane potential changes using JC-1 as a dye and Calcein/PI staining of different formulations in 4T1, MDA MB 231 and MDA MB 468 cell lines were characterized by Gen5 Cytation Imager. The students’ T test was used for statistical analysis.

Results: The DQ-Cu nanoparticles were prepared by a thin film dispersion method. The formed DQ and DQ-Cu nanoparticles had similar particle sizes around 200 nm in diameter. To confirm oxidative condition is the driver of the transition from DQ to DTC, we performed a calcein fluorescence competition assay. H₂O₂ was used to provide the oxidative condition. The fluorescence of calcein was quenched by the binding of copper ions, as shown in Cu/H₂O₂ and Cu/DQ groups in figure. After 15 min incubation, instant fluorescence recovery in Cu/NaDTC/H₂O₂ group was observed, indicating excessive DTC could chelate with Cu and released calcein from binding rapidly. In contrast, the Cu/DQ/H2O2 group showed a time-dependent fluorescence recovery. After around 2 h, both the Cu/NaDTC/H₂O₂ group and the Cu/DQ/H₂O₂ group showed similar calcein intensity. This indicated that under oxidative condition, DQ could be triggered into DTC by H₂O₂ which could then chelate with Cu. The MTT results showed only DQ-Cu nanoparticles exert great cytotoxicity in 4T1 , MDA MB 231 and MDA MB 468 cell lines, which indicated codelivery of copper ions with DQ was necessary. The JC-1 staining and Calcein AM/PI staining showed consistent results with the MTT assay.

Conclusion: We developed a novel codelivery system of DQ and Cu for cascade trigger-release, featuring an in situ DTC prodrug response and cancer treatment with tumor specificity. The BPEI was repurposed here to deliver copper ions into tumor with non-toxicity. The benefits of DQ-Cu nanoparticles go far beyond high cell toxicity, it also includes codelivery of DSF based prodrug and copper ions in one system with specific tumor selectivity. This work shows that DQ-Cu nanoparticles have the potential to disrupt intracellular reactive oxygen species (ROS) equilibrium and achieve in situ copper ion chelation. Therefore, our study provides a new technique that can be seen as a solution for DSF-based delivery systems with adequate in situ copper ion supply and exerted excellent cytotoxicity in 4T1, MDA MB 231 and MDA MB 468 cell lines.

References: [1] Q. Pan et al., “A dithiocarbamate-based H2O2-responsive prodrug for combinational chemotherapy and oxidative stress amplification therapy,” Chem. Commun., vol. 55, no. 92, pp. 13896–13899, 2019, doi: 10.1039/c9cc05438c.

Acknowledgements: This project was supported by Auburn University start-up fund (F. Li).

Figure 1. The nanoparticle sizes of different formulations by dynamic light scanning.

Figure 2. The serum stability of DQ-Cu nanoparticles by dynamic light scanning.

Figure 3. The Calcein AM/PI staining of 4T1 cells treated with different formulations.