C₃N nanodots inhibits Aβ peptides aggregation pathogenic path in Alzheimer's disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive and behavioral decline. A large body of evidence implicates aggregation of Aβ peptides—particularly Aβ₄₂ and certain N-truncated forms—as central to synaptic dysfunction, neuroinflammation, oxidative stress, tau hyperphosphorylation, and neuronal death. Aβ oligomers also disrupt membranes, receptors, signaling, mitochondria, Golgi and ER function, endolysosomal integrity, and autophagy. While reversing Aβ aggregation is a promising therapeutic strategy, few effective anti-aggregation treatments have succeeded clinically. Only a small number of antibody therapies (e.g., aducanumab, lecanemab; donanemab showing promise) target amyloid, and other agents (peptides, polymers, small molecules, metal oxides) generally show modest effects. Nanomaterials have emerged as candidates to modulate protein aggregation, but translation in vivo remains limited. This study investigates whether C₃N nanodots can inhibit Aβ aggregation, disassemble fibrils, cross the blood–brain barrier (BBB), and ameliorate AD pathology and behavioral deficits in APP/PS1 mice, while maintaining biocompatibility.

Prior work highlights limited success of anti-amyloid therapies, with only a few monoclonal antibodies (aducanumab, lecanemab, and promising data for donanemab) gaining traction. Other anti-AD agents including peptides, polymers, small molecules (e.g., EGCG, curcumin, indole-3-propionic acid, ferulic acid), and metal oxides have typically exhibited mild inhibition of Aβ aggregation. Nanomaterials such as graphene oxide, fullerenes, quantum dots, carbon nanotubes, and g-C₃N₄ have shown in vitro inhibition of amyloid fibrillization and the ability to disaggregate mature fibers; graphene quantum dots have also demonstrated inhibition of α-synuclein aggregation and BBB penetration in Parkinson’s disease models. The inhibitory efficacy of nanomaterials depends on physicochemical properties (size, curvature, surface chemistry), and concerns remain regarding in vivo performance and potential long-term toxicity for certain materials (e.g., GO-related inflammation). This context motivates exploring C₃N nanodots as potentially effective and biocompatible anti-aggregation agents in vivo.

• Synthesis and characterization: C₃N nanodots synthesized via hydrothermal polymerization of 2,3-diaminophenazine (320 °C, 36 h), oxidized with H₂O₂, and purified by dialysis. Characterized by TEM/HRTEM (lateral size ~4.5 ± 0.4 nm, lattice spacing 0.21 nm; height <1 nm), UV–Vis, FTIR, and XPS.
• In vitro aggregation assays: Aβ₄₂, AβpE3, and Aβ₄₀ monomer preparation via HFIP/DMSO protocols. Aggregation inhibition and disaggregation evaluated by Thioflavin-T (ThT) fluorescence kinetics, dot blot (mOC87, amyloid fibril-specific), AFM, TEM, and circular dichroism (CD) spectroscopy.
• Molecular dynamics simulations: All-atom MD (GROMACS, AMBER99SB-ILDN, TIP3P) of Aβ₄₂ dimers ± C₃N nanodot (diameter ~4.5 nm) to assess secondary structure evolution and interactions (van der Waals, electrostatics, hydrophobic, π–π stacking, hydrogen bonding). Comparative simulations with stacked C₃N, graphene (GRA), and C₆₀ fullerene evaluated inhibitory differences.
• Cell studies: Primary mouse cortical neurons, PC12, primary rat astrocytes, HUVECs, SH-SY5Y, bEnd.3, and BV2 cells used to assess cytotoxicity (CCK-8), LDH release, Live/Dead staining, and neuronal morphology by SEM. Assessed whether C₃N nanodots alleviate Aβ₄₂ oligomer-induced toxicity under co-incubation at varying nanodot concentrations.
• BBB penetration and biodistribution: Cy5.5-labeled C₃N (C₃N-Cy5.5) administered intraperitoneally (i.p.) to C57BL/6J mice at 200 mg/kg for ex vivo brain fluorescence imaging at 8 h, 24 h, 48 h, 1 week. Biodistribution quantified by IVIS; excretion tracked via metabolic cage collections after 100 mg/kg dosing.
• In vivo efficacy: Male APP/PS1 mice received daily i.p. injections of C₃N nanodots (1 or 5 mg/kg) or saline from 3 to 9 months of age. Behavioral assessments included Morris water maze (MWM; 5-day acquisition, probe on day 6) and novel object recognition (NOR). Post-treatment, brains analyzed for amyloid burden (6E10 immunostaining of cortex and hippocampus; quantification of plaque area and counts across size bins), and Aβ₄₂/Aβ₄₀ levels in TBS-, SDS-, and FA-soluble fractions by ELISA.
• Synaptic markers: Western blot for SNAP25 and VAMP2; immunofluorescence double labeling (MAP2 and SNAP25) to assess neuronal and synaptic integrity.
• Safety/toxicology: Body weight monitoring; H&E histology of heart, liver, spleen, lung, kidney; inflammation markers (WBC, Lymph#, Mon#, Gran#); liver and kidney function (AST, ALB, UREA); cytocompatibility comparisons vs GO nanosheets. Biodegradation assessed under lysosome-mimicking acidic conditions and catalase/H₂O₂ environments; cellular colocalization to lysosomes via confocal microscopy.
• Statistics: Student’s t tests, one-way or two-way ANOVA with appropriate post hoc tests; significance at p < 0.05.

• C₃N nanodot properties: Ultra-small lateral size 4.5 ± 0.4 nm; height <1 nm; high polarity due to C–N bonds and charged edge groups (–COO−, –NH₃⁺), favoring water dispersity and biomembrane compatibility.
• In vitro inhibition of aggregation: C₃N nanodots dose-dependently delayed Aβ₄₂ aggregation kinetics and reduced endpoint ThT fluorescence; dot blots (mOC87) showed reduced amyloid fibril content with higher C₃N concentrations; AFM/TEM revealed morphology shift from long mature fibrils to diffuse puncta. Similar inhibitory effects observed for AβpE3 and Aβ₄₀.
• Disaggregation of mature fibrils: Co-incubation of preformed Aβ₄₂ fibrils with C₃N led to time- and dose-dependent dismantling into smaller amorphous species (ThT, dot blot, CD, AFM, end-to-end distance analyses).
• MD simulations: In controls, Aβ₄₂ dimers formed partial β-sheets. With C₃N, β-sheet formation was strongly suppressed; overall β-sheet content dropped from about 10.6 ± 1.5% (without C₃N) to 0.2 ± 0.6% (with C₃N), with increases in random coil/bend components. Peptide adsorption to C₃N driven by dominant van der Waals plus electrostatic, hydrophobic, hydrogen-bonding, and π–π interactions; aromatic residues (F4, F20) exhibited π–π stacking; charged residues engaged with edge groups. Compared to nanographite and C₆₀, C₃N showed stronger inhibitory effects attributable to its polar surface and ability to form multiple interaction types.
• Cellular effects and cytocompatibility: Aβ₄₂ aggregation significantly reduced neuron survival (CCK-8), while C₃N nanodots dose-dependently improved neuron viability (e.g., survival increased across 100–500 µg/mL) and reduced LDH release; Live/Dead assays showed fewer dead cells. SEM indicated that C₃N preserved neuronal morphology and neurites against Aβ-induced damage. C₃N alone showed mild cytotoxicity even at 500 µg/mL and demonstrated broad cytocompatibility across multiple cell lines; superior biocompatibility vs graphene oxide nanosheets.
• BBB penetration: After i.p. administration of C₃N-Cy5.5 (200 mg/kg), brain fluorescence was detectable by 8 h, peaked at 48 h, and declined to undetectable by 1 week, indicating BBB crossing and brain accumulation.
• Behavioral improvements in APP/PS1 mice: Daily i.p. C₃N for 6 months shortened MWM escape latency vs saline. A 1 mg/kg/day dose outperformed 5 mg/kg/day on day 5 (mean latencies approximately 19.2 s vs 29.3 s), with no differences in swimming speed. In probe trials, C₃N-treated mice spent more time in the target quadrant (about 15.9 s vs 6.5 s for APP/PS1) and crossed the platform location more frequently (about 7.2 vs 3.0). Novel object recognition improved to levels comparable to WT (recognition index near 0.7 for treated APP/PS1).
• Amyloid pathology reduction: 6E10 immunostaining showed marked decreases in plaque area in cortex and hippocampus after 1 mg/kg/day C₃N (plaque area reduced by ~60%). ELISA of cortical fractions showed total Aβ₄₂/Aβ₄₀ reductions by ~36%/~50%, with the largest decreases in FA-soluble dense-plaque forms (~84%/~83%), indicating strong inhibition of dense plaque formation.
• Synaptic markers: Western blots showed increases in SNAP25 (~43%) and VAMP2 (~22%) after C₃N treatment. MAP2-positive neuron counts and SNAP25/MAP2 co-localization were significantly higher in treated mice, indicating synaptic protection.
• Safety and biodegradation: Body weights increased steadily; H&E of major organs revealed no lesions; inflammatory indices (WBC, Lymph#, Mon#, Gran#) remained in normal ranges; liver/kidney function tests (AST, ALB, UREA) unchanged. Biodistribution indicated liver and kidney as primary off-target organs. Excretion occurred via urine and feces. C₃N showed degradability in lysosome-like acidic conditions and catalase/H₂O₂ environments; confocal imaging confirmed lysosomal localization, supporting intracellular degradation.

The study demonstrates that C₃N nanodots effectively inhibit Aβ peptide aggregation and disassemble mature fibrils, shifting Aβ self-assembly away from β-sheet-rich structures toward disordered, off-pathway species. This molecular action reduces Aβ-induced neuronal toxicity in vitro and translates in vivo to reduced amyloid burden, restored synaptic protein levels, and significant improvements in learning and memory in APP/PS1 mice. MD simulations elucidate the mechanistic basis—dominant van der Waals interactions complemented by electrostatic, hydrophobic, π–π, and hydrogen bonding—underlying peptide adsorption to C₃N and disruption of β-structure formation. Compared to nanographite and fullerene, C₃N’s polar surface and charged edge groups enable stronger, multifaceted interactions with Aβ, correlating with superior anti-aggregation efficacy and better dispersion/biocompatibility. The nanodots cross the BBB after systemic administration and exhibit favorable biodistribution, minimal systemic toxicity, and indications of biodegradability, supporting their potential as a safe anti-amyloid nanotherapeutic platform. Collectively, these findings address the central hypothesis that a rationally designed carbon nitride nanodot can modulate Aβ aggregation pathways and ameliorate AD-related pathology and behavior in vivo.

C₃N nanodots are potent inhibitors of Aβ aggregation that can also disassemble mature fibrils, reduce neuronal toxicity, penetrate the BBB, lessen amyloid plaque burden (especially dense plaques), preserve synaptic integrity, and improve cognitive performance in APP/PS1 mice, all with minimal observed toxicity and evidence of biodegradability. These results provide experimental and computational validation for C₃N nanodots as a promising nanomaterial-based therapeutic strategy against AD. Future work should optimize nanodot surface chemistry and dosing, evaluate pharmacokinetics and long-term safety, assess efficacy in female mice and additional AD models, explore effects on tau pathology and neuroinflammation, and advance towards translational studies.

• Sex and model limitations: Only male APP/PS1 mice were used; findings may not generalize to females or other AD models.
• Dosing and concentration: Cellular assays used relatively high peptide and nanodot concentrations; imaging required high systemic doses (200 mg/kg) to visualize brain signals.
• Long-term safety and biodistribution: Although no major toxicity was observed, long-term accumulation in off-target organs (liver, kidney) and chronic effects require further investigation.
• Mechanistic scope: The study focuses on Aβ; effects on tau pathology, neuroinflammation, and broader neural circuitry were not deeply characterized.
• Sample sizes: Some in vivo analyses involved modest cohort sizes (e.g., n=3 for certain biochemical/imaging endpoints), warranting larger confirmatory studies.