An In Situ Chemotherapy Drug Combined with Immune Checkpoint Inhibitor for Chemoimmunotherapy

Abstract

Clinically, cancer chemotherapy still faces unsatisfactory efficacy due to drug resistance and severe side effects, including tiredness, hair loss, feeling sick, etc. The clinical benefits of checkpoint inhibitors have revived hope for cancer immunotherapy, but the objective response rate of immune checkpoint inhibitors remains around 10-40%. Herein, two types of copper-doped mesoporous silica nanoparticles (MS-Cu-1 with a diameter of about 30 nm and MS-Cu-2 with a diameter of about 200 nm) were synthesized using a one-pot method. Both MS-Cu-1 and MS-Cu-2 nanoparticles showed excellent tumor microenvironment regulation properties with elevated extracellular and intracellular ROS generation, extracellular and intracellular oxygenation, and intracellular GSH depletion. In particular, MS-Cu-2 nanoparticles demonstrated a better microenvironment modulation effect than MS-Cu-1 nanoparticles. The DSF/MS-Cu composites with disulfiram (DSF) and copper co-delivery characteristics were prepared by a straightforward method using chloroform as the solvent. Cell survival rate and live/dead staining results showed that DSF and MS-Cu alone were not toxic to LLC cells, while a low dose of DSF/MS-Cu (1-10 μg/mL) showed a strong cell-killing effect. In addition, MS-Cu-2 nanoparticles released more Cu²⁺ in a weakly acidic environment (pH = 5) than in a physiological environment (pH = 7.4), and the Cu²⁺ released was 41.72 ± 0.96 mg/L in 1 h under weakly acidic conditions. UV-visible absorption spectrometry confirmed the production of tumor-killing drugs (CuETs). The intratumoral injection of DSF/MS-Cu significantly inhibited tumor growth in vivo by converting nontoxic DSF/MS-Cu into toxic CuETs. The combination of DSF/MS-Cu and anti-CTLA-4 antibody further inhibited tumor growth, showing the synergistic effect of DSF/MS-Cu and immune checkpoint inhibitors.

Keywords: chemoimmunotherapy; copper-dopped mesoporous silica (MS-Cu); disulfiram (DSF); nontoxic to toxic; tumor environment regulation.

Figure 1

Scheme of MS-Cu synthesis (A), TEM images (B), particle size distribution of MSCu–1 (C) and MS-Cu–2 (D), Cu/Si mol ratios (E), N₂ adsorption–desorption isotherms (F), BET surface areas (G), and zeta potential (H) of MS-Cu–1 and MS-Cu–2 nanoparticles (*** p < 0.001).

Figure 2

Cytotoxicity (A) and live/dead staining (B) of DSF/MS-Cu–1 and DSF/MS-Cu–2 (*** p < 0.001).

Figure 3

Extracellular ROS generation (A,B), intracellular ROS generation (C), and fluorescence images of intracellular ROS level in LLC cells (D) induced by MS-Cu–1 and MS-Cu–-2 nanoparticles (*** p < 0.001).

Figure 4

Extracellular oxygenation (A) and intracellular oxygenation (B) ability, and fluorescence images of intracellular oxygenation level in LLC cells stained with RDPP (C) (** p < 0.01, *** p < 0.001).

Figure 5

Intracellular relative GSH/GSSG content in LLC cells incubated with MS-Cu–1 and MS-Cu–2 nanoparticles (* p < 0.5, ** p < 0.01).

Figure 6

Ion release (A) of MS-Cu–2 nanoparticles soaked in PBS (pH = 7.4), ion release (B) of MS-Cu–2 nanoparticles soaked in acetate buffer (pH = 5.0), and UV-vis absorption (C) of CuET, MS-Cu–2, and DSF/MS-Cu–2 in ethanol.

Figure 7

Experimental protocol (A), body weight (B), tumor volume (C), and tumor weight (D) (* p < 0.5, ** p < 0.01, and *** p < 0.001).

Scheme 1

Synergistic anti-tumor efficacy of DSF/MS-Cu and immune checkpoint inhibitors.