Carbon dots as dual inhibitors of tau and amyloid-beta aggregation for the treatment of Alzheimer's disease

Abstract

Alzheimer's disease (AD) is the most common form of senile dementia, presenting a significant challenge for the development of effective treatments. AD is characterized by extracellular amyloid plaques and intraneuronal neurofibrillary tangles. Therefore, targeting both hallmarks through inhibition of amyloid beta (Aβ) and tau aggregation presents a promising approach for drug development. Carbon dots (CD), with their high biocompatibility, minimal cytotoxicity, and blood-brain barrier (BBB) permeability, have emerged as promising drug nanocarriers. Congo red, an azo dye, has gathered significant attention for inhibiting amyloid-beta and tau aggregation. However, Congo red's inability to cross the BBB limits its potential to be used as a drug candidate for central nervous system (CNS) diseases. Furthermore, current studies only focus on using Congo red to target single disease hallmarks, without investigating dual inhibition capabilities. In this study, we synthesized Congo red-derived CD (CRCD) by using Congo red and citric acid as precursors, resulting in three variants, CRCD1, CRCD2 and CRCD3, based on different mass ratios of precursors. CRCD2 and CRCD3 exhibited sustained low cytotoxicity, and CRCD3 demonstrated the ability to traverse the BBB in a zebrafish model. Moreover, thioflavin T (ThT) aggregation assays and AFM imaging revealed CRCD as potent inhibitors against both tau and Aβ aggregation. Notably, CRCD1 emerged as the most robust inhibitor, displaying IC₅₀ values of 0.2 ± 0.1 and 2.1 ± 0.5 µg/mL against tau and Aβ aggregation, respectively. Our findings underscore the dual inhibitory role of CRCD against tau and Aβ aggregation, showcasing effective BBB penetration and positioning CRCD as potential nanodrugs and nanocarriers for the CNS. Hence, CRCD-based compounds represent a promising candidate in the realm of multi-functional AD therapeutics, offering an innovative formulation component for future developments in this area. STATEMENT OF SIGNIFICANCE: This article reports Congo red-derived carbon dots (CRCD) as dual inhibitors of tau and amyloid-beta (Aβ) aggregation for the treatment of Alzheimer's disease (AD). The CRCD are biocompatible and show strong fluorescence, high stability, the ability to cross the blood-brain barrier, and the function of addressing two major pathological features of AD.

Keywords: Alzheimer's disease; Blood-brain barrier; Carbon dots; Congo red; Dual inhibitors.

Fig. 1.

(A) UV/vis absorption spectrum of CRCD1, CRCD2 and CRCD3 aqueous solutions. (B-D) Fluorescence emission spectra of CRCD1, CRCD2 and CRCD3 in aqueous medium, respectively.

Fig. 2.

FTIR spectrum of CRCD1 (black), CRCD2 (red) and CRCD3 (green).

Fig. 3.

TGA spectra of (A) CRCD1, (B) CRCD2 and (C) CRCD3; DTG spectra of (D) CRCD1, (E) CRCD2 and (F) CRCD3.

Fig. 4.

XPS spectra of C 1s of (A) CRCD1, (B) CRCD2, (C) CRCD3; O 1s of (D) CRCD1, (E) CRCD2, (F) CRCD3; N 1s of (G) CRCD1, (H) CRCD2, (I) CRCD3; S 2p of (J) CRCD1, (K) CRCD2 and (L) CRCD3. XPS background was corrected using a Shirley line-shape type function.

Fig. 5.

Mass spectra of (A) CRCD1; (B) CRCD2 and (C) CRCD3. All samples were dispersed in DI water at a concentration of 0.1 mg/mL.

Fig. 6.

AFM images with size distribution of CRCD1, CRCD2 and CRCD3.

Fig. 7.

Cytotoxicity measurements of CRCD1, CRCD2, CRCD3 and Congo red at different concentrations that were applied to (A) HeLa and (B) HEK 293 cells and incubated for 72 hrs. The cell viability of the control group was shown as 100%. Data are exhibited as the mean ± STD. Statistical significance was calculated using a t-test for 100 μg/mL condition in comparison with the 0 μg/mL control group and indicated as no significance (ns) (p-value > 0.05) and ** (p-value < 0.01).

Fig. 8.

ThT fluorescence data demonstrating (A) dose dependence of all three CRCD on tau aggregation and (B) dose dependence of all three CRCD on amyloid-beta aggregation. Statistical significance was calculated using a t-test for all three in comparison with the uninhibited control and indicated as * (p-value < 0.05), ** (p-value < 0.01), and *** (p-value < 0.001).

Fig. 9.

Tau aggregation can be visualized via Atomic Force Microscopy, demonstrated above. Uninhibited tau can be seen farthest to the left with extensive fibril formation compared with the image on the far right with a 143 μg/mL of CRCD1, demonstrating minimal aggregation and confirming the presence of CRCD1.

Fig. 10.

Tau aggregation can be visualized via Transmission Electron Microscopy, demonstrated above. Uninhibited tau can be seen farthest to the right with extensive fibril formation compared with the image on the far left with 143 μg/mL (240μM) CRCD1, demonstrating minimal aggregation and confirming the presence of CRCD1.

Fig. 11.

The IC₅₀ values and standard deviations calculated from the ThT fluorescence of four replicates suggests CRCD1 is most effective at inhibiting tau and Aβ42 aggregation, seemingly more effective against tau (A) than Aβ42 (B). Statistical significance was calculated using a t-test for all three in comparison with the uninhibited control and indicated as * (p-value < 0.05), ** (p-value < 0.01), and *** (p-value < 0.001).

Fig. 12.

Simplified schematic interpretation of CRCD interference with β-sheet aggregation.

Fig. 13.

Confocal images of zebrafish larvae injected with CRCD1, CRCD2 and CRCD3 aqueous solution (30 mg/mL) in contrast to the control group. The red arrow indicates the central canal of spinal cord of zebrafish. Each picture was reproduced with at least six larvae.