Nanoparticle characterisation through dynamic light scattering measurements. The hydrodynamic diameter increase of single- (ANP:PNA, green curve) and double- (ANP:PNA:NGF, blue curve) functionalised polyacrylamide nanoparticles resulted in a shift of size distribution compared to naked ANPs (red curve).

Nanoparticle distribution through retinal layers after intravitreal injection in zebrafish larvae. (a) Representative images of a retinal cross-section of a zebrafish larva IVT injected with Alexa Fluor 488-positive polyacrylamide nanoparticles, analysed at 4 h post-injection (hpi). Hoechst staining allows identification of the retinal layers. In FITC channel, the intense green spots clearly highlight the presence of Alexa Fluor 488-positive nanoparticles in the injected eye. (b) Quantitative evaluation of the distribution of Alexa Fluor 488-positive polyacrylamide nanoparticles in the retinal layers at different time points. n = 15 injected eyes from 3 independent experiments. GCL, ganglion cell layer; INL, inner nuclear layer; PR, photoreceptors; RPE, retinal pigment epithelium. Scale bar: 50 µm.

Prolonged localisation of nanoparticles at the site of injection. Representative images of 3 days post-fertilisation (dpf) larvae IVT injected with naked Alexa Fluor 594-positive nanoparticles (ANPs) or FITC-labelled PNA-conjugated polyacrylamide nanoparticles (ANP:PNA), analysed at 4 and 24 h post-injection (hpi). As control, zebrafish larvae were IVT injected with DPBS. White asterisk indicates the pronephros. n = 45 injected eyes from 3 independent experiments. Scale bar: 300 µm.

NGF exerts its biological activity upon nanoformulation. (a) Representative images of PC12 cells incubated for 4 days with 100 ng/mL of free NGF or with the same concentration of NGF bound to PNA-conjugated polyacrylamide nanoparticles (ANP:PNA:NGF). As control, PC12 cells were incubated with PNA-conjugated nanoparticles (ANP:PNA) or DPBS. Red and blue asterisks indicate the presence of neurites in cells exposed to NGF and ANP:PNA:NGF, respectively. Scale bar: 300 µm. (b) Quantitative evaluation of neurite length in PC12 cells incubated with NGF or ANP:PNA:NGF. n = 180 neurites from 3 biological replicates, t-test for unpaired data followed by Kolmogorov–Smirnov test. (c) Protective effect of NGF-conjugated polyacrylamide nanoparticles against oxidative stress. ARPE-19 cells were cotreated with 100 ng/mL of free NGF (NGF) or nanoformulated NGF (ANP:PNA:NGF) and 300 µM of H₂O₂ for 24 h. MTT assay was performed to evaluate cell viability. Absorbance values of all the groups were normalised to those of the untreated control sample (CTRL), which was set at 100%. n = 3 biological replicates, one-way ANOVA applying Tukey’s multiple comparisons test. (b,c) ns, p > 0.05 and ****, p < 0.0001.

Nanoformulated NGF improves visual function impaired by oxidative stress in zebrafish larvae. (a) Experimental timeline relative to the intravitreal injection of free NGF or NGF bound to PNA-conjugated polyacrylamide nanoparticles (ANP:PNA:NGF) in zebrafish larvae at 4 days post-fertilisation (dpf), followed by H₂O₂ exposure to induce oxidative stress in the retina. As control, zebrafish larvae were IVT injected with DPBS. (b) Evaluation of visual function by OKR assay in zebrafish larvae exposed to the treatments shown in panel (a). n ≥ 39 larvae for each group, Kruskal–Wallis followed by Dunn’s multiple comparisons test. (c) Quantitative evaluation of apoptosis through the detection of active caspase 3 (Casp3)-positive cells by immunofluorescence in zebrafish larvae exposed to the treatments shown in panel (a). n ≥ 20 larvae for each group, one-way ANOVA using Dunnett’s multiple comparisons test. (b,c) ns, p > 0.05; *, p < 0.01; ***, p < 0.001 and ****, p < 0.0001.