Fig 1: BDNF levels and associated transcriptional profiles in rabbit ocular tissues at 72 hours and 28 days following nanoparticle administration. (A) BDNF concentrations in the vitreous body and (B) retina of rabbits’ eyes measured at 72 hours and 28 days after administration of vehicle (Ringer), HSA-PEG NPs, or nanoparticle formulations (NeO10, NeO100). Data are presented as mean ± SD (n = 3 biological replicates per group). (C) Heatmap representation of relative mRNA expression levels of selected rabbit genes associated with MAPK/p38 signaling (Cnksr2, Nkap, Mknk1, Mapkapk2, Mapkapk3), AKT/mTOR axis (Akt1, Mtor) CREB- (Crebbp) and NFκB-related (Mknk1) transcriptional regulation, and proliferation/apoptosis (Pcna, Bcl2, Casp3, and Tp53). Gene expression was quantified by RT-qPCR using the 2–ΔΔCt method, with intact control samples serving as calibrator (set to 1). Values were log2-transformed prior to visualization to allow symmetric representation of relative up- and downregulation (log2 fold change relative to control). Samples were analyzed at early (72 h) and late (28 days) time points following treatment with Ringer, NeO10, or NeO100. The color scale indicates relative expression changes compared with intact control, with red tones representing higher relative expression and blue tones representing lower relative expression. Each column represents the mean expression value per group (n = 3 biological replicates). Given the limited sample size, no formal inferential statistical testing was performed; the analysis is presented as exploratory and descriptive, focusing on directional trends and relative magnitude of change rather than statistical significance. Individual sample-level expression values are provided in Supplementary Figure 1 to illustrate inter-sample variability.
Fig 2: Physicochemical characterization of PEGylated HSA-NT3-BDNF nanoparticles over 28 days (40,320 min) after formulation. Particle size distributions were measured by intensity (A) NeO 10; (B) NeO 5. (C) ζ -potential was measured in Ringer’s solution (open symbols: NeO 5; filled symbols: NeO 10). Particle concentrations for NeO 10 and NeO 5 were determined by MADLS (D). All syntheses were performed in six replicates, and error bars denote mean ± SD. Individual points represent NP diameter distributions obtained from eight samples.
Fig 3: AFM characterization of PEGylated HSA-NT3-BDNF nanoparticle diameter distribution over 28 days after formulation. AFM images of nanoparticles deposited from Ringer’s solution at pH 7.0 ± 0.6 are shown 24 h (A) and 28 days (C) after formulation, with corresponding X-axis cross-sections (B and D) acquired over a 20×20 µm area. Notably, the dispersed morphology of PEGylated NT3-BDNF nanoparticles was preserved over 28 days (40,320 min).
Fig 4: Cross-context biological validation of PEGylated HSA nanoparticles co-loaded with BDNF and NT3. Schematic overview of the study workflow and key biological findings. (A) PEGylated human serum albumin (HSA) nanoparticles were formulated for dual delivery of BDNF and NT3. (B) Cross-context validation included physicochemical characterization (MADLS/DLS, zeta potential/ELS, AFM), in vivo rabbit eye assessment following intravitreal administration, and in vitro oxidative stress models using ARPE-19 retinal pigment epithelial cells and RA-differentiated SH-SY5Y dopaminergic-like neurons. (C) Functional analyses demonstrated intracellular neurotrophin delivery associated with improved mitochondrial membrane potential, reduced apoptosis, decreased lipid peroxidation, and supportive transcript-level responses under oxidative stress conditions. (D) The downstream panel highlights canonical PI3K/AKT/mTOR and RAS/RAF/MEK/ERK signaling axes induced by NT3/BDNF that may contribute to CREB-associated stress-protective responses. The schematic illustrates a conceptual downstream signaling framework and does not imply a specific receptor-mediated nanoparticle uptake mechanism. Conceptual workflow illustration created with assistance from ChatGPT (OpenAI, GPT-5 image generation tools) and finalized by the authors.
Fig 5: Assessment of MDA concentration in ARPE19 cell supernatant after 48 hours. ARPE19 cells were exposed to 10 mM NaIO3, NeO5, NeO10, HSA-PEG or BDNF/NT3 (10 mg L−1). MDA levels were normalized to the total protein concentration in each supernatant sample. Data presented as mean±SEM from at least three biological replicates. Statistical significance was determined using the one-way ANOVA: ***p < 0.001, **p < 0.01, *p < 0.05. Red stars are significance in relation to control.
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