Delivery of an immunogenic cell death-inducing copper complex to cancer stem cells using polymeric nanoparticles

The major cause for cancer related deaths worldwide is tumour relapse and metastasis, both of which have been heavily linked to the existence of cancer stem cells (CSCs). CSCs are able to escape current treatment regimens, reform tumours, and promote their spread to secondary sites. Recently, our research group reported the first metal-based agent 1 (a copper(ii) compound ligated by a bidentate 4,7-diphenyl-1,10-phenanthroline and a tridentate Schiff base ligand) to potently kill CSCs via cytotoxic and immunogenic mechanisms. Here we show that encapsulation of 1 by polymeric nanoparticles at the appropriate feed (10%, 1 NP10) enhances CSC uptake and improves potency towards bulk cancer cells and CSCs (grown in monolayer and three-dimensional cultures). The nanoparticle formulation triggers a similar cellular response to the payload, which bodes well for further translation. Specifically, the nanoparticle formulation elevates intracellular reactive oxygen species levels, induces ER stress, and evokes damage-associated molecular patterns consistent with immunogenic cell death. To the best of our knowledge, this is the first study to demonstrate that polymeric nanoparticles can be used to effectively deliver immunogenic metal complexes into CSCs.


Fig. S1
UV-Vis spectrum of 1 (25 μM) in DMSO over the course of 24 h at 37 o C.  ESI mass spectra (positive mode) of 1 (500 μM) in H 2 O:DMSO (10:1) (A), and in the presence of ascorbic acid (5 mM) (B) or in the presence of glutathione (5 mM) (C) after incubation for 24 h at 37 o C.

Fig. S9
Dynamic light scattering size distribution of 1 NP 10 suspended in water. Size refers to diameter of nanoparticles in nm.

Fig. S10
Dynamic light scattering size distribution of empty PEG-PLGA nanoparticles suspended in water. Size refers to diameter of nanoparticles in nm.
Electronic Supplementary Material (ESI) for RSC Advances. This journal is © The Royal Society of Chemistry 2022

Fig. S11
Variation in 1 NP 10 diameter upon incubation in water, PBS with 10% FBS, and mammary epithelial growth medium (MEGM) over the course of 72 h at 37 o C.

Fig. S13
Copper content in HMLER and HMLER-shEcad cells treated with 1 NP 10 (110 nM for 4 h) at 4 o C or 37 o C.

Fig. S14
Copper content in HMLER-shEcad cells treated with 1 NP 10 only (16 nM for 24 h), and upon pre-incubation with ammonium chloride (50 mM for 2 h) or chloroquine (100 μM for 2 h) at 37 o C. Error bars represent standard deviations and Student t test, * = p < 0.05.

Fig. S15
The amount of copper released from 1 NP 10 upon incubation in PBS (pH 7.4) or sodium acetate buffer (pH 5.2) over the course of 72 h at 37 o C.

Fig. S16
Representative dose-response curves for the treatment of HMLER and HMLER-shEcad cells with 1 NP 10 .

Fig. S17
Representative dose-response curves for the treatment of HMLER and HMLER-shEcad cells with empty PEG-PLGA nanoparticles.

Fig. S18
Representative bright-field images (× 10) of HMLER-shEcad mammospheres in the absence and presence of salinomycin at its respective IC 20 values for 5 days.

Fig. S20
Representative dose-response curve for the treatment of HMLER-shEcad mammospheres with empty PEG-PLGA nanoparticles.

Fig. S21
Representative dose-response curves for the treatment of HMLER-shEcad cells with 1 NP 10 after 72 h incubation in the presence of salubrinal (10 µM).

Fig. S22
Immunoblotting analysis of proteins related to the unfolded protein response (UPR). Protein expression in HMLER-shEcad cells following treatment with 1 NP 10 (40-191 nM) for (A) 4 h or (B) 24 h.

Fig. S24
Representative dose-response curves for the treatment of HMLER-shEcad cells with 1 NP 10 after 72 h incubation in the presence of z-VAD-FMK (5 µM).