Gadolinium metallofullerenol nanoparticles inhibit cancer metastasis through matrix metalloproteinase inhibition: imprisoning instead of poisoning cancer cells

Synthesis and characterization of Gd@C₈₂(OH)₂₂ nanoparticles (f-NPs). (A) The hydroxyl-functionalized Gd@C₈₂ nanoparticles were purified by Sephadex G-25 column chromatography with an eluent of distilled water. TEM image shows the morphology of f-NPs. Scale bar, 50 nm. (B) Size distributions, average sizes (first value in parentheses), and zeta potentials of f-NPs (second value in parentheses) in different physiological aqueous solutions were analyzed. (C) Stability of f-NPs in physiological solution was determined by transparency check, DLS, and ICP-MS analysis. f-NPs were suspended in saline with or without mouse serum (w/w, 1%) at 100 μg/mL and sonicated for 10 minutes before the measurement. Particle size measurements were conducted using DLS from 0 to 6 days. Photographs of f-NPs in saline were taken at 0 and 6 days. To determine whether Gd³⁺ ions could be released from the carbon cage, dialysis experiments were performed. The dialysis bags contained 5 mL f-NPs suspension at particle dose of 100 μg/mL, which was dialyzed against 200 mL saline with or without serum. Gd content in dialysis bags were measured at 0 and 6 days by ICP-MS.

Study of the effects of f-NPs on MMP regulation and cancer cell invasion in vitro. (A) qPCR (histogram) and western blotting (insert) were used to study the level of MMP-9 and MMP-2 in U937 cells treated with f-NPs at various concentrations. ^⁎P < 0.05, compared to control. (B) qPCR (histogram) and western blotting (insert) were used to determine MMP-9 and MMP-2 levels in MDA-MB-231/U937 co-cultured system in the presence of f-NPs at indicated concentrations. ^⁎P < 0.05, compared to control. (C) To study the effect of f-NPs on cancer cell invasion, the GFP-labeled cancer cells that were able to pass through the Matrigel-coated membranes toward serum-containing medium (chemoattractant) were detected. PMA-, caffeine-, and small-molecule MMP inhibitors (CP 471474, CID 10667540)–treated cells were included in the assay. Nontreated cells were considered as 100% in the calculation of the invasion index. ^⁎P < 0.05, significantly lower compared to control; #P < 0.05, significantly higher compared to non-treated cells.

The f-NPs potently inhibited tumor metastasis in the tissue invasion animal model. (A) Representative bioluminescence images (BLI) of tumor foci obtained from control (left) and the f-NPs–treated animals (right) after the course of treatment. (B) Quantification of the bioluminescence intensity of tumor foci in the lungs of animals after the course of treatment. f-NPs treatment significantly inhibited tumor metastasis with an R_M = 78% in the tissue invasion model. ^⁎P < 0.05, compared with saline control. (C) The levels of MMP-9 and MMP-2 in the primary tumor tissues treated with f-NPs or saline were determined by qPCR (histogram) and western blotting (insert). ^⁎P < 0.05, compared to control.

Photomicrographs of histological slides of excised tumors using Van Gieson staining to differentiate collagen fibers at tumor site in control and f-NPs–treated animals, respectively. (A) The excised tumors of the control mice and (B) of the f-NPs–treated mice of MDA-MB-231 human breast cancer model. (C) The excised tumors of the control mice and (D) of the f-NPs–treated mice of another human breast cancer model, MCF-7. The dotted line represents the boundary between the tumor and its fibrous capsule. The average thicknesses of fibrous capsules on the tumors in the f-NPs–treated group were 7–18 times larger than those of the control depending on tumor type.

f-NPs potently inhibited the establishment of tumor foci in the blood transfer model of MDA-MB-231-luc cancer mice. (A) MDA-MB-231-luc cells were harvested and resuspended at a concentration of 1 × 10⁷ cells/mL in saline. A 0.1-mL cell suspension was injected into the tail vein of the nude mice. Seven days after cancer cell injection, the mice received daily intraperitoneal doses of the f-NPs at 2.5 μmol/kg for a duration of 6 weeks. Saline was used as control. Tumor metastases in lung were monitored weekly by BLI. (B) Quantification of the BLI intensity of tumor foci in the lungs of animals after different treatments. f-NPs treatment significantly inhibited tumor metastasis with an R_M = 88% in the bloodstream transfer model. ^⁎P < 0.05, compared to control. (C) Histological examination of the lungs with different treatments was performed. Arrows indicate the tumor foci in a saline-treated animal. Higher magnification images of animal lung are shown in the lower panel.

Schematic presentation of possible antimetastasis mechanism of f-NPs. Rather than direct cell killing, the metallofullerenol nanoparticles inhibited tumor metastases mainly through a MMP inhibition process. In the control group (left panel), TAMs-secreted MMP enzymes are able to efficiently degrade the fibrous matrices surrounding tumors and facilitate their invasiveness. In the nanoparticle-treated group (right panel), f-NPs decreased the production of MMP, subsequently reducing fibrous matrix degradation. The thick fibrous cage may therefore serve as a “prison” that tightly confines the invasive cancer cells within the primary site.