Reactive oxygen species activated by mitochondria-specific camptothecin prodrug for enhanced chemotherapy

Abstract

Camptothecin (CPT) has attracted much attention due to its potent antitumor activities. However, the undesirable physicochemical properties, including poor water-solubility, unstable lactone ring and severe adverse effects limit its further application. In this study, two water-soluble prodrugs, CPT-lysine (CPTK) and CPT-arginine (CPTR), were designed and synthesized by conjugating lysine or arginine with CPT, improving its solubility, pharmacokinetic properties and tumor penetration. Importantly, the introduction of arginine into CPTR contributed to the mitochondria-specific delivery, which increased mitochondrial reactive oxygen species (ROS) generation, induced mitochondria dysfunction and enhanced cell apoptosis and in vivo anti-cancer effect. This strategy is believed to hold great potential for organelle-specific synergistic anti-tumor therapy.

SCHEME 1

Schematic illustration of the anticancer mechanism of CPT prodrugs. CPT: camptothecin.

FIGURE 1

Characterization of CPTK/FBS and CPTR/FBS. (A) Particle size of CPTK and CPTR with different CPT concentration in FBS (10%); (B) zeta potential; and (C and D) the SEM picture of CPTR/FBS and CPTK/FBS. *** p < 0.001 (Student's t-test).

FIGURE 2

In vitro cytotoxicity of CPT, CPTK, and CPTR in different cell lines. (A) HeLa cells; (B) MCF7 cells; (C) CT26 cells; (D) 4T1 cells; and (E) B16F10 cells. Cell viability was evaluated by MTT assay after different treatments for 48 hours.

FIGURE 3

Cellular uptake in CT26 cell, CPTK, and CPTR labeled with FITC (6 hours, 5 μM).

FIGURE 4

Measurement of apoptosis rates of CT26 cells treated with CPT, CPTK, or CPTR and the ROS detection. (A) Flow cytometry analysis of apoptosis (24 hours, 5 μM); (B) apoptosis rate (24 hours, 5 μM); and (C) ROS generation after CPT, CPTR, and CPTK treatment (6 hours, 5 μM). The statistical significance was calculated through one-way ANOVA test. **p < 0.01.

FIGURE 5

CLSM images of colocalization of mitochondria and CPT (6 hours, 5 μM). Blue color represents CPT, CPTK, or CPTR. Mitochondria were stained with MitoTracker Red (red).

FIGURE 6

Variations in mitochondrial and calcium homeostasis. (A) CLSM images of mitochondrial potential changes (2 hours, 5 μM); (B) flow cytometry analysis of mitochondrial membrane potential changes (2 hours, 5 μM); and (C) evaluation of calcium ion concentration (24 hours, 5 μM). *p < 0.05, ***p < 0.001, ****p < 0.0001 (one-way ANOVA).

FIGURE 7

In vitro tumor penetration of CPT, CPTK, and CPTR in 3DSMs after 2 or 24 hours of incubation (with the CPT concentration of 10 μg/mL). Scale bar represents 200 μm.

FIGURE 8

In vivo anti-tumor therapy. (A) CPT concentration in blood versus time after intravenous injection of CPT, CPTK, and CPTR in mice (10 mg/Kg of CPT concentration); (B) tumor volume changes during treatment (0, 3, 6, 9, and 12 were the time point of drug injection); (C) the body weight changes during treatment; (D) tumors were excised from each group after the sacrifice of mice; and (E) H&E staining images of tumor collected from CPTK and CPTR injected mice and control treated mice with PBS. ** p < 0.01 (one-way ANOVA).

FIGURE S1

Synthesis and characterization of CPTR and CPTK. (A) Synthesis procedures of CPTR and CPTK; (B) their structures characterized by 1H-NMR spectra; and (C) mass spectrometer. *CPTR was synthesized as our previous work [51].

FIGURE S2

(A and B) Stability of CPTR and CPTK measured by fluorescence spectrum and (C, D and E) MTT analysis in A549, B16F10, and CT26 cells.

FIGURE S3

(A and B) UV spectrum of CPTK and CPTR in various solvent (CPT concentration: 10 μM).

FIGURE S4

ROS detection in 4T1, B16F10, and HeLa cells (Bright field, green fluorescence) (5 μM).

FIGURE S5

H&E staining images of heart, liver, lung, spleen, kidney, and tumor collected from CPTK and CPTR injected mice and control treated mice with PBS.