A Chemical structure of the copolymers employed to prepare NPs. B Representative Fixed aqueous layer thickness (FALT) measurements evaluated by monitoring the zeta potential of NPs dispersed in NaCl solutions in water ([NPs] = 250 μg/mL): the slope of the straight line represents the thickness of the outer hydrophilic shell expressed in nm. C Docetaxel (DTX) release from NPs in PBS 10 mM pH 7.4, at 37 °C evaluated by the dialysis method. Results are the mean values ± SD of three measurements carried out on three different NPs batches

A Hydrodynamic diameter of NPs after incubation in PBS pH 7.4 at 37 °C at different time points ([NPs] = 1 mg/mL). B Percentage of folate exposure on NP surface measured by incubating NPs (5 mg/mL) with a monoclonal anti-folic acid antibody. Results are the mean values ± SD of three measurements carried out on three different NPs batches. C Fluorescence emission spectra of RPMI with 10% of FBS at Ex = 278 nm in the presence of DBL NPs, DBL_Fol NPs and DBL_Fol/aFLT1 ([NPs] = 1 mg/mL). The reduction of the emission of RPMI + FBS indicates an interaction between NPs and the protein

Confocal images of KB spheroids after treatment with DiO-loaded NPs (50 µg/mL) for 48 h: a bright-field; b DiO fluorescence acquired in the equatorial plane of the spheroid; c 3D reconstruction of the distribution of the fluorescence signal in the equatorial plane of the spheroid; d maximum projection obtained by superimposing the images of the 20 acquired focal planes. Scale bars: 100 μm

Cytotoxicity of DTX-loaded NPs in KB spheroids. A Percentage of residual ATP in the spheroid measured using the CellTiter-Glo® 3D Cell Viability Assay after 72 h of treatment with DTX-loaded NPs or with the free drug ([DTX] = 0.001–1 μg/mL). Data are mean values ± SD of at least three independent experiments carried out in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 vs. free DTX (Student’s t-test). B Bright-field images of spheroids acquired by confocal microscopy after 72 h of treatment with DTX-loaded NPs at different doses. Scale bars: 100 μm

Toxicity and activity of DTX-loaded NPs in the zebrafish embryo xenograft model. A Timeline of the in vivo experiments. The microscopy image shows 3 embryos xenografted with KB cells (red fluorescence). B Toxicity of NPs in xenograft zebrafish embryos injected with DTX and DTX-loaded NPs ([DTX] = 2.5 ng/animal) after 6 days from the treatment. C Tumor volume reduction after 6 days from the treatment with DTX-loaded NPs ([DTX] = 2.5 ng/animal). Total tumor fluorescence quantified from images. ^§p < 0.05; ^§§p < 0.01; ^§§§p < 0.001 vs. ctrl; *p < 0.001 vs. free DTX. D Representative images of KB cells xenotransplanted in the embryo yolks. Scale bars: 500 μm

Vasculature analysis of fli1a:EGFP embryos after two days of treatment with nanoparticles (NPs) ([DTX] = 2.5 ng/animal). Fluorescence microscopy images of untreated embryos without (A) or with KB xenografted cells (B); embryos with KB xenografted cells and treated with free DTX (C) or DTX-DBL_Fol/aFLT1 (D). B1 is a magnification showing the presence of vascular branches (white circles) sprouting toward tumor masses in untreated embryos. In the magnification, D1 vascular branches are not visible due to the anti-angiogenic effect exerted by DTX-DBL_Fol/aFLT1. Scale bar: 250 μm. Red: tumor cells stained with DiI; green: zebrafish embryo vasculature