(M0930-05-28) Disulfiram-Loaded Metal-Organic Nanoparticles Trigger Immunogenic Cell Death of the Triple-Negative Breast Cancer

Purpose: Disulfiram (DSF), initially used for treating alcohol dependency, has been the focus of recent research to repurpose as a cancer therapeutic alone or to enhance therapies for drug-resistant cancers. However, the DSF-copper complex (Cu(DDC)₂) is water-insoluble, which limits its clinical potential. The primary goal of this study is to employ Stabilized Metal Ion Ligand Complex (SMILE) technology for the preparation of Cu(DDC)₂ nanoparticles (NPs). Then, we characterized the efficacy and underlying mechanism of the Cu(DDC)₂ NPs inhibiting cancer cells.

Methods: In this study, PEG-Lysine dendrimer-Cholic Acid (PLCA) polymers were synthesized. In brief, PEG-NH₂ was conjugated with Fmoc-protected lysine followed by deprotection of fmoc to expose the free amine group, which was reacted with NHS-ester of cholic acid to generate PLCA. The nanoparticle size distribution was characterized using a Zetasizer instrument. The concentration of Cu(DDC)₂ within the nanoparticles was determined by an UV-Vis absorbance. The efficacy of the Cu(DDC)₂ NPs was evaluated by MTT assays and immunofluorescence staining.

Results: The synthesized dendrimers demonstrated significant improvements in the stability and drug loading of Cu(DDC)₂ NPs. We analyzed different ratios of cholic acid to PEG. We found that the PEG-lysine-cholic acid₈ (PLCA₈) achieved a very high drug loading percentage (approximate 100%) and the particle size was around 50 nm. Additionally, PLCA8 formed Cu(DDC)₂ NPs with excellent serum stability.Then, we selected the PLCA8 as a stabilizer to prepare the Cu(DDC)₂ NPs. We conducted an MTT assay to determine the anticancer activity of the Cu(DDC)₂ NPs, which exhibited potent anticancer activity (with an IC₅₀ of 230 nM). On the other hand, DSF, CuCl₂, DDCNa, and the PLCA8 polymer did not show apparent toxicity on 4T1 cells. When we evaluated the underlying mechanism of the Cu(DDC)₂ NPs anticancer activity, we observed an increase in calreticulin (CRT) and heat shock protein expression, and the adenosine triphosphate (ATP) was also released from the cancer cells. These markers support that the Cu(DDC)₂ NPs can trigger the immunogenic cell death. Furthermore, Cu(DDC)₂ NPs also demonstrated significant inhibition of in vivo tumor growth, reducing tumor size by 40%.

Conclusion: We synthesized amphiphilic dendrimers to prepare Cu(DDC)₂ NPs. With excellent loading percentage of resultant nanoparticles with a mean particle size of 50 nm. We anticipate increased tumor accumulation due to the enhanced permeability and retention (EPR) effect. The Cu(DDC)₂ NPs exhibited potent anticancer activity in both in vitro and in vivo. We further demonstrated that the CRT was translocated to the cell surface, ATP was released from the cells and the HSP expression increased, all of which are important markers for immunogenic cell death (ICD). Triggering ICD may enhance the infiltration of cytotoxic T cells and facilitate the maturation of the dendritic cells. Consequently, this will enable cell-mediated immunity to eliminate cancer cells. These findings suggest a promising pathway for further development of DSF-based nanoparticles as a novel anticancer agent.

References: 1. Chen, W.; Yang, W.; Chen, P.; Huang, Y.; Li, F., Disulfiram copper nanoparticles prepared with a stabilized metal ion ligand complex method for treating drug-resistant prostate cancers. ACS applied materials & interfaces 2018, 10 (48), 41118-41128.
2.Xiao, K.; Luo, J.; Fowler, W. L.; Li, Y.; Lee, J. S.; Xing, L.; Cheng, R. H.; Wang, L.; Lam, K. S., A self-assembling nanoparticle for paclitaxel delivery in ovarian cancer. Biomaterials 2009, 30 (30), 6006-6016.


Different numbers of Cholic acid show different properties. PLCA2 = PEG5k-Lysine-(Cholic acid)2; PLCA4 = PEG5k-(Lysine)2-(Cholic acid)4; PLCA8 = PEG5k-(Lysine)4-(Cholic acid)8 (A) Loading percentage (B) particle size (C) change of drug concentration and (D) particle size of Cu(DDC)2 NPs prepared with different numbers of cholic acid conjugated stabilizers.


4T1 cells induced ICD after treating with PBS (Control), Cu(DDC)2 (1 µM), and Cu(DDC)2 NPs (1 µM). The Cu(DDC)2 NPs were prepared with PLCA8 polymer. (A) Levels of intracellular ATP were analyzed by flow cytometry. (B) The level of the released ATP was analyzed by an ATP detection kit. (C) The level of the releasing HMGB1 was measured with an ELISA kit. (D) Levels of CRT expression were detected by flow cytometry.


Cu(DDC)2 NPs antitumor activities in vivo and in vitro. (A) 4T1 cells were treated with different formulations for 48 h and analyzed with the MTT assay. (B) Growth of 4T1 tumor in BALB/c mice injected with PBS (control) or Cu(DDC)2 NPs. (C) The measurement of tumor weight at the end of the study. (D) Change of body weight in 4T1 tumor model mice during the study.