Repurposing suramin for the treatment of breast cancer lung metastasis with glycol chitosan-based nanoparticles

Abstract

Suramin (SM), a drug for African sleeping sickness and river blindness therapy, has been investigated in various clinical trials for cancer therapy. However, SM was eventually withdrawn from the market because of its narrow therapeutic window and the side effects associated with multiple targets. In this work, we developed a simple but effective system based on a nontoxic dose of SM combined with a chemotherapeutic agent for the treatment of metastatic triple-negative breast cancer (TNBC). SM and glycol chitosan (GCS) formed nanogels because of the electrostatic effect, whereas doxorubicin (DOX) was incorporated into the system through the hydrophilic and hydrophobic interactions between DOX and GCS as well as the ionic interactions between DOX and SM to yield GCS-SM/DOX nanoparticles (NPs). GCS-SM/DOX NPs have a size of approximately 186 nm and a spherical morphology. In vitro experiments showed that GCS-SM NPs could effectively inhibit cancer cell migration and invasion, as well as angiogenesis. Furthermore, in a TNBC lung metastasis animal model, GCS-SM/DOX NPs significantly reduced tumor burden and extended the lifespan of animals, while not inducing cardio and renal toxicities associated with the DOX and SM, respectively. As all the components used in this system are biocompatible and easy for large-scale fabrication, the GCS-SM/DOX system is highly translatable for the metastatic breast cancer treatment. STATEMENT OF SIGNIFICANCE: The doxorubicin-loaded glycol chitosan-suramin nanoparticle (GCS-SM/DOX) is novel in the following aspects: SM acts as not only a gelator for the first time in the preparation of the nanoparticle but also an active pharmaceutical agent in the dosage form. GCS-SM/DOX NP significantly reduced tumor burden and extended the lifespan of animals with triple-negative breast cancer lung metastasis. GCS-SM/DOX NPs attenuate cardio and renal toxicities associated with the DOX and SM. The GCS-SM/DOX system is highly translatable because of its simple, one-pot, and easy-to-scale-up preparation protocol.

Keywords: Antiangiogenesis; Antimetastasis; Doxorubicin; Nanoparticle; Suramin; Triple-negative breast cancer.

Fig. 1.

Scheme of the assembly of GCS-SM/DOX NP and its action in treating breast cancer lung metastasis.

Fig. 2.

The effects of (A) SM amount (GCS and SM were dissolved in PBS, pH 7.0; SM concentration of 0.5 mg/ml), (B) pH of the buffer (with SM concentration of 0.5 mg/ml), (C) SM concentration, and (D) the loading content of DOX (in PBS, pH 7.0) on the hydrodynamic size (black) and PDI (red) of the nanoparticle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

Characterization of GCS-SM/DOX NP. (A) Hydrodynamic size distribution and surface charge (inserted). (B) Transmission electron microscopy images of GCS-SM/DOX NPs with 100 K magnification. Scale bar is 100 nm. (C) In vitro release of suramin and doxorubicin in PBS. (D) Hydrodynamic size of GCS-SM NPs when co-incubated with 10%–30% FBS medium. (E) Long-term stability GCS-SM NPs in PBS.

Fig. 4.

The effect of GCS-SM NP on the migration and invasion of MDA-MB-231 cells. (A) Images of cell migration of cells after treated with SM, GCS or GCS-SM NP at different concentrations at 0, 4, 8, 12, 16, 20 h. Scale bars are 150 μm. (B) Quantification of migration distance of each time point. (C) Average migration speed of each treatment groups. (D) Cytotoxicity of SM or GCS-SM NPs at different concentrations against MDA-MB-231 cells for 24 h. (E) Representative images of MDA-MB-231 cells in the invasion assay. Scale bars are 200 μm. (F) Cell counting result after 16 h of invasion study. (n = 5 pictures per treatment, #P < 0.01).

Fig. 5.

The in vitro HUVEC tube formation assay. (A) Photographs of representative tubes formed in different treatments. Magnification: 10×. Scale bar: 200 μm. (B) Confocal microscopy images of representative tubes formed in different treatments. Magnification: 100×. Scale bar: 50 μm. (C) Quantitative data for endothelial cell tube formation expressed as length of tubes/field ± Std. (D) Quantitative data for endothelial cell tube formation expressed as number of tubes/field ± Std. **P < 0.001 compared with control.

Fig. 6.

The uptake and cytotoxicity of GCS-SM/DOX nanoparticles and DOX for MDAMB-231 cells. (A) Confocal microscopy of the uptake of DOX or GCS-SM/DOX NP in MDAMB-231 cells for 3 h. Hoechst 33,342 (blue), DOX (red). Scale bars are 20 μm. (B) FACS spectra of MDA-MB-231 cells treated with free DOX or GCS-SM/DOX NP for 3 h or 6 h. (C) DOX distribution in the cytosol and nuclei after treating MDA-MB-231 with DOX or GCS-SM/DOX NP for 1 h. (D) The cytotoxicity of suramin and GCS-SM NP after 48 h of treatment. (E) Cell viability of DOX in combination with different concentrations of suramin in cells pretreated with a/b FGF. (F) Combination index (CI) of DOX and GCS-SM NPs mixed at different ratios. (G) Cell viability of combinational nanoparticles at different ratios. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7.

The inhibitory effect of GCS-SM/DOX NP on tumor growth in vivo using bioluminescence assay. (A) Cell luminescence intensity as a function of cell number. (B) Representative luminescence images of mice from different treatment groups from week 1 to week 6. (C) Mice body weight change curves over the experiment. GCS-SM/DOX NP increased survival rate and reduced side effects. (D) Timeline of the in vivo experiments. (E) Survive curves of different treatments. (F) Lung weight in different treatments. (*P < 0.05, **P < 0.01).

Fig. 8.

Histology analysis and TEM analysis of organs from different treatment groups. (A) H&E stained images of lungs, livers, and kidneys. Black arrows indicate the tumor (T) area in the lung. Green arrows indicate the glomerulonephritis areas in the kidney. (B) Transmittance electron microscopy images of heart tissues from the control, free drug combination, and GCS-SM/DOX NP treatment groups. Scale bars are 2 μm. (C) IHC detection of CD31 in tumor tissues of different treatment groups. IHC staining (CD31) of tumor blood vessels (brown), microvessels (red arrow). Scale bars are 50 μm. The tumors were cut into 5 μm thick sections, stained with CD31, and imaged (10 fields per section) (*P < 0.05 compared with control). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)