Rationally Designed Redox-Active Au(I) N-Heterocyclic Carbene: An Immunogenic Cell Death Inducer

Abstract

Immunogenic cell death (ICD) is a way of reengaging the tumor-specific immune system. ICD can be induced by treatment with chemotherapeutics. However, only a limited number of drugs and other treatment modalities have been shown to elicit the biomarker responses characteristic of ICD and to provide an anticancer benefit in vivo. Here, we report a rationally designed redox-active Au(I) bis-N-heterocyclic carbene that induces ICD both in vitro and in vivo. This work benefits from a synthetic pathway that allows for the facile preparation of asymmetric redox-active Au(I) bis-N-heterocyclic carbenes.

Figure 1.

(A) Synthetic scheme for 1. (a) NaOH (7.5 equiv), t-amyl alcohol (cat.), t-butyl alcohol (cat.), THF, RT, 24 h. (b) Acetone, RT, 4 h. c) toluene, 90 °C, 24 h. (B) ORTEP representation of 1 rendered using POV-Ray. Thermal ellipsoids are at the 50% probability level.

Figure 2.

Confocal microscopy images showing ROS generation in A549 cells 4 h post treatment with 1.6 μM of 1. In the case of 1, the merged image of Mitotracker Red and CM-H₂DCFDA provides evidence for ROS production in the mitochondria.

Figure 3.

(A) Confocal microscopy images showing calreticulin (CRT) translocation in CT26 cells 4 h post-treatment with 5 μM of 1. No CRT release was seen in the case of vehicle alone. (B) Flow cytometry showing dose dependent CRT release in CT26 cells 24 h post treatment with 1 or oxaliplatin. (C) Dose dependent ATP release in CT26 cells 4 h post-treatment with 1 or oxaliplatin. (D) Dose dependent release of HMGB1 protein in CT26 cells 4 h post-treatment with 1 or oxaliplatin (one-way ANOVA with Tukey’s multiple comparison). Data are mean ± SD and p < 0.05 is significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 4.

(A) Schematic diagram illustrating in vivo strategies. (B) Percent tumor-free mice (i.e., left flank tumor) injected with pretreated CT26 cells with varying doses of 1 or 150 μM oxaliplatin in the right flank and then inoculated (i.e., challenged) with CT26 cells in the left flank when right flank tumors were not resected (n = 10, data were combined from two independent experiments). (C) Percent tumor-free mice (i.e., left flank tumor) when right flank tumors were resected before they reached >200 mm³ in size. Mice (n = 10) were censored (i.e., removed from the study) if they reached an end point for euthanasia before developing a left flank tumor (for details, see Supporting Information). (Mantel-Cox Log-rank test, p < 0.005 (Bonferroni-corrected) was considered significant, * indicates difference from freeze–thaw.)