Synergistic Antioxidant and Anti‑Amyloid Action of Chlorogenic Acid–Selenium Nanoparticles in Vitro

Abstract

Accumulation of amyloid‑β (Aβ) plaques and reactive oxygen species (ROS) underpins Alzheimer’s disease (AD) pathology. Here we report a novel nanocomposite—chlorogenic acid–coated selenium nanoparticles (CGA@SeNPs)—that combines the Aβ‑binding capacity of SeNPs with the antioxidant potency of chlorogenic acid (CGA). In vitro assays reveal that while free CGA scavenges Aβ‑induced ROS, it fails to block Aβ aggregation or protect neuronal membranes. Conversely, CGA@SeNPs markedly inhibit Aβ40 fibrillogenesis, reduce ROS generation, and preserve PC12 cell viability and membrane integrity, outperforming CGA alone. These findings highlight CGA@SeNPs as a promising dual‑target therapeutic candidate for AD.

Background

Alzheimer’s disease (AD) is a relentlessly progressive neurodegenerative disorder marked by cognitive decline and neuronal loss. A defining hallmark of AD is the extracellular deposition of misfolded amyloid‑β (Aβ) plaques, which not only disrupts synaptic function but also catalyzes ROS production, driving oxidative damage to DNA, lipids, and proteins. The neurotoxic Aβ oligomers and protofibrils are increasingly recognized as primary culprits in AD pathogenesis.

Nanoparticles (NPs) offer unique physicochemical attributes—small size, large surface area, and tunable reactivity—that enable them to interact with Aβ, modulate its aggregation, and deliver therapeutic agents. Selenium nanoparticles (SeNPs) are especially attractive due to their biocompatibility, low toxicity, and essential role in cellular redox homeostasis. Prior studies have demonstrated that surface modification of SeNPs with anti‑amyloid peptides can enhance their sequestration of Aβ. However, the impact of conjugating natural antioxidants to SeNPs on both aggregation inhibition and ROS scavenging remains unexplored.

Chlorogenic acid (CGA) is a polyphenol abundantly present in coffee and various fruits. It exhibits potent anti‑oxidative, anti‑inflammatory, and neuroprotective effects, yet its clinical utility is limited by poor bioavailability. By grafting CGA onto SeNPs, we aim to overcome these limitations and evaluate the composite’s therapeutic potential against Aβ‑mediated toxicity.

Methods and Experimental Design

Materials and Cell Lines

Aβ40 peptide, selenium dioxide, sodium borohydride, MTT, Thioflavine T, DCFH‑DA, and other reagents were sourced from Sigma. CGA was obtained from Aladdin. PC12 cells were cultured in DMEM supplemented with 5% FBS and 10% horse serum at 37 °C, 5% CO₂.

Preparation of CGA@SeNPs

SeNPs were synthesized by reducing a CGA–sodium selenite mixture with NaBH₄. Optimal CGA:SeO₂ ratio was 1:6, yielding ~100 nm spherical particles as confirmed by TEM.

Characterization

• Transmission electron microscopy (TEM) for morphology.
• Energy‑dispersive X‑ray spectroscopy (EDX) for elemental composition.
• UV‑vis and fluorescence spectroscopy for optical properties.
• Dynamic light scattering (DLS) for size stability in PBS.

Antioxidant and ROS Assays

Hydroxyl, ABTS⁺, and superoxide scavenging activities were quantified using commercial kits. Aβ‑induced H₂O₂ production was measured with DCFH‑DA fluorescence.

Aβ Aggregation Monitoring

Thioflavine T fluorescence tracked fibril formation over 5 days. TEM visualized fibril morphology. Resonance light scattering (RLS) assessed binding affinity between CGA@SeNPs and Aβ monomers.

Cytotoxicity and Apoptosis Evaluation

MTT and LDH release assays measured cell viability and membrane integrity, respectively. Intracellular ROS was detected by DCFH‑DA, while TUNEL‑DAPI staining identified apoptotic nuclei in PC12 cells exposed to Aβ aggregates ± CGA or CGA@SeNPs.

Results and Discussion

Characterization of CGA@SeNPs

TEM images showed uniform, ~100 nm spheres. EDX confirmed Se (30 %) and characteristic CGA signals (C 52.6 %, O 1.5 %). UV‑vis absorption shifted from 334 nm (free CGA) to 337 nm upon conjugation, while fluorescence emission red‑shifted from 443 nm to 438 nm, confirming surface attachment. DLS data demonstrated stable particle size in PBS over 7 days.

Enhanced Antioxidant Activity

CGA@SeNPs exhibited dose‑dependent scavenging of hydroxyl, ABTS⁺, and superoxide radicals, achieving 70 %–95 % inhibition at 60 µg/mL—superior to CGA or vitamin C. In Aβ40‑induced H₂O₂ assays, CGA@SeNPs reduced ROS generation more effectively than free CGA, underscoring synergistic antioxidant capacity.

Inhibition of Aβ Aggregation

Thioflavine T fluorescence revealed that CGA@SeNPs dose‑dependently suppressed Aβ40 fibril formation, reducing peak fluorescence by ~50 % at 60 µg/mL. TEM corroborated these findings: no fibrils were observed in the presence of CGA@SeNPs, whereas CGA alone did not alter fibril morphology. RLS data indicated strong binding of CGA@SeNPs to Aβ monomers, likely sterically hindering nucleation.

Neuroprotective Effects

In PC12 cells, Aβ40 reduced viability to 53 %. CGA@SeNPs restored viability to ~95 %, whereas CGA only improved it to 66 %. LDH release mirrored these trends, with CGA@SeNPs normalizing membrane integrity. Intracellular ROS and apoptosis assays (DCFH‑DA, TUNEL) demonstrated that CGA@SeNPs attenuated Aβ‑induced oxidative stress and DNA fragmentation, while CGA offered partial protection.

Conclusions

Chlorogenic acid conjugated to selenium nanoparticles endows the composite with dual anti‑amyloid and antioxidant functions. CGA@SeNPs bind Aβ, prevent fibril assembly, scavenge ROS, and safeguard neuronal cells from membrane damage and apoptosis. Compared with free CGA, the nanocomposite offers enhanced efficacy and stability, positioning it as a promising therapeutic strategy for mitigating AD pathology.

Abbreviations

ABTS⁺

2, 29‑Azinobis‑(3‑ethylbenzothiazoline‑6‑sulfonic acid)

AD

Alzheimer’s disease

Aβ

Amyloid‑β

CGA

Chlorogenic acid

DCFH‑DA

2′,7′‑Dichlorodihydrofluorescein diacetate

LDH

Lactate dehydrogenase

MTT

Thiazolyl blue tetrazolium bromide

ROS

Reactive oxygen species

SeNPs

Selenium nanoparticles

TEM

Transmission electron microscope

ThT

Thioflavine T

TUNEL

Terminal transferase dUTP nick end labeling