Alzheimer's disease (AD) is a prevalent neurodegenerative disease with no cure. Toxic amyloid-beta (Aβ) plaques and harmful inflammation are hallmarks of AD, exacerbating each other. Current AD treatments are mono-targeted, lack specificity, have poor blood-brain barrier (BBB) penetration, and offer low imaging sensitivity. The development of a visual therapeutic drug with BBB penetration, high specificity, and dual-targeting potential is urgently needed. High-sensitivity imaging, particularly fluorescence imaging in the near-infrared-II (NIR-II) region (1000–1700 nm), offers advantages for in vivo monitoring and drug design. Existing probes have limitations in BBB penetration, short emission wavelengths, aggregation-caused quenching (ACQ), poor Aβ affinity, and lack of therapeutic activity. This study aims to develop a therapeutic-type NIR-II probe with aggregation-induced emission (AIE) properties to visualize the BBB-traversing and Aβ plaque binding processes, while simultaneously providing dual-targeted therapy for AD.

The existing literature highlights the significant unmet need for effective Alzheimer's disease treatment. Current therapies, such as acetylcholinesterase inhibitors, NMDA receptor antagonists, anti-inflammatory agents, and monoclonal antibodies, are limited by their mono-targeted approach, low specificity, poor BBB penetration, and potential toxicities. The lack of effective dual-targeting therapies that address both Aβ plaques and neuroinflammation underscores the importance of developing novel therapeutic strategies. While several in vitro probes for Aβ plaques exist, they lack the necessary properties for in vivo imaging and therapeutic application, including poor BBB penetration, short emission wavelengths, and the absence of therapeutic functionality. The challenge of developing an ideal visual drug with superior NIR-II emission, strong Aβ affinity, and dual-targeting therapeutic capabilities, has remained unmet until this study.

This research synthesized two therapeutic-type NIR-II AIEgens, compound 3 and compound 6. Compound 3 contains 2,2′-azanediylbis(ethan-1-ol) groups for Aβ fibril binding, while compound 6 incorporates terpyridyl coordinated Ce(III) units for antioxidant capability. These AIEgens were co-assembled into reactive oxygen species (ROS)-responsive nanocomposites (Ang-AIE NCs or Ang-NCs) using a thioketal (TK)-containing template and brain-targeted angiopep-2 (Ang-2) modification. The characterization included NMR, HRMS, FMO analysis, aggregation-induced PL studies, DLS, TEM, FTIR, and NIR imaging. The ROS-responsive release of the AIEgens from the NCs was evaluated using DLS and other techniques. The anti-inflammatory, fibril disassembly, and inhibition of fibril generation properties were assessed using various assays, including oxygen generation assays, ESR spectroscopy, X-ray photoelectron spectroscopy, TEM, and DLS. Molecular dynamics (MD) simulations were conducted to explore the detailed interaction mechanism of compound 3 with Aβ fibrils. In vitro studies included cytotoxicity assays, BBB penetration assessment using a Transwell model, confocal imaging (ThT, DCFH-DA, MitoSOX, JC-1), RT-PCR, ELISA, and Western blotting to evaluate the neuroprotective effects. In vivo studies were performed on female APP/PS1 transgenic AD mice. Ang-NCs were intravenously administered, and in vivo NIR-II imaging, pharmacokinetic analysis, ROS detection, immunofluorescence staining (Aβ, Iba-1, GFAP), Prussian Blue histochemistry, behavioral assessments (nest construction, Morris water maze, novel object recognition), and safety assessments (H&E staining, blood parameters) were conducted. Statistical analysis included one-way ANOVA and two-tailed Student's t-tests.

The study successfully synthesized and characterized two NIR-II AIEgens, compound 3 and compound 6. Compound 3 exhibited strong Aβ fibril binding affinity (Kₐ = 2.6 µM) through Van der Waals forces, hydrogen bonding, and π-π interactions, as confirmed by MD simulations and DFT calculations. Compound 6, containing Ce(III), demonstrated effective ROS scavenging capabilities. Co-assembly of these AIEgens into Ang-NCs resulted in a nanotheranostic with enhanced hydrophilicity, longer in vivo circulation half-life (4.8 h), improved BBB penetration, and strong NIR-II emission at 1350 nm, enabling high-sensitivity in vivo imaging. In vitro, Ang-NCs exhibited no significant cytotoxicity, effectively degraded Aβ fibrils, scavenged ROS, alleviated inflammation (reduced TNF-α, IL-1β, IL-6 mRNA and protein levels), and protected cells from Aβ-induced toxicity. In vivo, Ang-NCs showed significant BBB penetration, reduced ROS levels, decreased Aβ plaques, reduced inflammation markers, and improved behavioral and cognitive functions in AD mice (as evidenced by the nest construction, Morris water maze, and novel object recognition tests). Importantly, the treatment did not cause cerebral microhemorrhage. The results demonstrated the synergistic effects of the two AIEgens in a self-enhanced therapy program.

The findings address the significant unmet clinical need for effective dual-targeting therapy of AD. The successful development and evaluation of Ang-NCs, a novel NIR-II nanotheranostic, provide a significant advancement in the field. The dual-targeting strategy, targeting both Aβ plaques and neuroinflammation, along with the high-sensitivity NIR-II imaging capability, represents a major step forward compared to current mono-targeted therapies. The study demonstrates that a combined approach targeting both Aβ plaques and inflammation significantly improves therapeutic efficacy. The successful in vivo delivery and therapeutic effects of Ang-NCs showcase the potential of this technology for translating into clinical practice. The findings also suggest guidelines for future designs of NIR-II nanotheranostics for other neurodegenerative diseases.

This study successfully developed and validated a novel NIR-II nanotheranostic system (Ang-NCs) for the dual-targeting treatment of Alzheimer's disease. The Ang-NCs achieved effective BBB penetration, high-sensitivity in vivo imaging, targeted Aβ fibril degradation, and inflammation suppression, resulting in significant cognitive and behavioral improvements in an AD mouse model. This research provides a promising therapeutic strategy for AD and a valuable template for designing advanced NIR-II nanotheranostics for other neurodegenerative diseases. Future research could explore further optimization of the nanotheranostic formulation, exploring other neurodegenerative diseases, and conducting larger-scale preclinical studies.

While this study provides strong evidence for the efficacy of Ang-NCs in a mouse model of AD, several limitations warrant consideration. The study used a female AD mouse model; the results may not be fully generalizable to males or humans. The long-term effects of Ang-NCs remain to be investigated. Further research is needed to fully elucidate the mechanism of action and optimize the formulation for human use. The sample sizes in some of the in vivo experiments could be considered relatively small for definitive conclusions.