Fig 1.
Illustration of the multi-targeting concept shows a dual-ligand nanoparticle targeting the dynamic nature of metastatic disease.
Fig 2.
Targeting EGFR and αvβ3 integrin in the D2.A1 mouse model of metastasis.
(a) Bioluminescence imaging (BLI) shows the development of metastasis in the lungs (left). FMT in vivo imaging was performed 3 h after injection of a cocktail of EGFR-NP and RGD-NP. Using the different NIR fluorophores (Vivotag 680 and 750) on each nanoparticle variant, the fluorescence signal in each metastatic site of the FMT images was quantified for each formulation (n = 5 mice). On the basis of phantom measurements of each formulation using the FMT system, the fluorescence signal was converted to nanoparticle concentration. (b) In a different animal study, a cocktail of EGFR-NP and RGD-NP labeled with a different fluorophore (Alexa 647 and 750) was intravenously injected into animals with D2.A1 metastasis. After 3h from injection, lungs were perfused, excised, sectioned into thin slices of equal thickness and imaged ex vivo using an IVIS Spectrum system. The signal from each lung slice was quantified and summarized for the entire lung indicating the total number of each nanoparticle variant in the lungs of different mice with metastasis (n = 4 mice).
Fig 3.
Treatment of mice with D2.A1 metastasis using dual-ligand nanoparticle loaded with doxorubicin.
(a) The cytotoxicity of doxorubicin (DOX) was evaluated on D2.A1 cells. Cytotoxicity studies were performed by seeding D2.A1 cells at a density of 5 x 103 cells per well. Cells were incubated with the treatment for 24 h at a concentration ranging between 0.2–1 μM DOX. After treatment application, the cells were washed three times with fresh medium and then incubated for 48 h at 37°C. The number of viable cells was determined using a formazan-based cell counting assay (CCK-8). Untreated cells served as live controls for normalization of the data. Data points represent group mean ± s.d. (b) The timeline and schedule of treatments are shown with respect to tumor inoculation. (c) The response of cancer metastasis to treatment was monitored using longitudinal BLI imaging. Quantification of the BLI signal in the thoracic region is shown for mice with D2.A1 metastasis treated at days 3, 4 and 5. In addition to untreated animals, treatments included non-targeted NP (NT-NP), RGD-NP, EGFR-NP, dual-ligand NP, and free DOX (n = 6–8 mice per treatment). The y-axis is in logarithmic scale. All nanoparticle formulations were administered at 7.5 mg/kg DOX (two-way ANOVA with repeated measures).
Fig 4.
The survival time of metastasis-bearing mice treated with cytotoxic drugs was compared to untreated animals.
The animals were treated at days 3, 4 and 5. The dual-ligand nanoparticle formulation was administered at 7.5 mg/kg DOX. In addition to the nanoparticle formulation, treatments included free DOX injected at 7.5 or 2.5 mg/kg (n = 6–8 mice per treatment). The difference between the survival curves of the dual-ligand NP and EGFR-NP-treated groups was assessed by the log-rank (Mantel-Cox) test.
Fig 5.
Histological evaluation of the microdistribution of RGD-NP and EGFR-NP nanoparticles in metastasis in the lungs of mice.
(a) Representative fluorescence image of lung tissue shows dispersion D2.A1 metastatic cancer cells (top; 20X magnification). After 3 h from injection of a cocktail containing Alexa 350-labeled EGFR-NP and Alexa 568-labeled RGD-NP, the two targeting variants colocalized in locations with metastatic cancer cells (bottom). Different regions with metastatic cancer cells were predominantly targeted by (b) EGFR-NP or (c) RGD-NP (green: D2.A1 cancer cells; red: RGD-NP; blue: EGFR-NP). (d) A pixel-by-pixel quantification indicates individual events for EGFR and RGD-NP or their overlap (n = 3, grouped analysis ANOVA; correct for multiple comparisons using the Holm−Sidak method. P values: *0.024, **0.002).