Nitrogen doped carbon quantum dots: a multifaceted carbon nanomaterial that interferes in an amyloid-forming trajectory

Abstract

Carbon quantum dots (CQDs) are a versatile class of carbon-based nanomaterial frameworks that have previously been used as a diagnostic device, in sensing for environmental applications, in bioimaging, and for drug delivery systems. Their versatility stems from their ability to be chemically tailored via functionalization to optimize properties for specific applications. In this study, we have synthesized lactic acid-derived nitrogen doped carbon quantum dots (LAdN-CQDs) and examined their ability to intervene in the conversion of soluble, monomeric hen egg-white lysozyme (HEWL) into mature fibrils. Our data indicate that LAdN-CQDs inhibit HEWL fibril formation in a dose-dependent manner (achieving up to 50% inhibition at 2.5 mg mL⁻¹). Furthermore, in a neuroblastoma-derived cell line, LAdN-CQDs were found not to disrupt mitochondrial membrane potential or trigger apoptosis at the same concentration range, suggesting that they are biocompatible. LAdN-CQDs effectively neutralized reactive oxygen species (ROS), with a 50% decrease in ROS levels at just 100 μg mL⁻¹ when challenged with an established free radical generator and protected the cell line from rotenone-induced apoptosis. The ability of LadN-CQDs to inhibit the soluble-to-toxic transformation of HEWL, the tolerance of SHSY-5Y cells to LAdN-CQDs, and their ability to restitute cells from rotenone-induced apoptosis, combined with the biocompatibility findings, suggest that LAdN-CQDs are potentially neuroprotective. The findings indicate that LAdN-CQDs represent a versatile, carbon-based, sustainable nanoplatform that bridges nanotechnology and neuroprotection, promoting the development of green chemistry-based healthcare solutions.

---

Introduction

Aging continues to be the predominant risk factor for diseases associated with protein misfolding, such as Alzheimer's disease (AD), Parkinson's disease (PD), systemic lysozyme amyloidosis, and Huntington's disease.^1–8 Common features of these aforementioned neurodegenerative disorders include increased levels of oxidative stress, mitochondrial dysbiosis, and the transformation of monomeric proteins like α-synuclein (in PD) and amyloid beta (in AD) into insoluble aggregates. These processes lead to neuronal damage and the loss of dopaminergic neurons in PD and cholinergic neurons in AD.^9,10

Carbon quantum dots (CQDs) have emerged as key alternatives to existing strategies such as natural product-based scaffolds to prevent the onset and progress of neurodegenerative disorders. CQDs possess sp²-hybridized carbon centers which are suitable for scavenging free radicals. Furthermore, their ability to be chemically tuned to cross the blood–brain barrier makes them particularly attractive for applications related to neuroprotection. Doping of nitrogen into CQDs enhances its antioxidant efficiency and adds new functionalities to the CQD surface, such as active linking groups for facile molecular conjugation. Such advances have led to superior performance across a suite of applications compared to undoped CQDs, making lactic acid-derived nitrogen-doped CQDs (LAdN-CQDs) a focal point of global research in recent years, particularly in the multifunctional nanomedicine domain.^11,12 Although LAdN-CQDs are in the early stages of development, scientists are motivated to further innovate their design and expand their functions and applications in environmental, signaling, and biomedical applications.^11–15 Furthermore, their abundant availability and simple synthesis using the commonly available hydrothermal method qualifies them as sustainably produced, and compatible with green-chemistry.^16–18 Contrary to small molecules, LAdN-CQDs enable easy modification of their chemical structures, which is crucial for the development of next-generation quantum dots designed through structure–activity relationship analyses.^19,20

Previous work has demonstrated that CQDs can intervene in neurodegenerative trajectories. In a rodent model of PD, graphene quantum dots were found to inhibit the soluble-to-toxic fibril formation of α-synuclein and provoke the disaggregation of mature fibrils.^1,19,20 Other work has demonstrated that carboxylic-acid- and gelatin-derived quantum dots are able to reset elevated levels of reactive oxygen species (ROS) generated in cell lines upon neurotoxicant exposure. These are important features because the buildup of misfolded protein aggregates and increased oxidative stress independently and synergistically contribute to the progress of neurodegeneration.^21–24 Proteins prone to aggregation, including Aβ, HEWL, islet amyloid precursor protein (IAPP), α-synuclein, and mutant Huntingtin protein (mHTT), transition from a soluble-state to toxic-aggregates during which their monomeric forms aggregate into mature fibrils.¹⁶ During this process, monomeric proteins undergo dimerization and oligomerization, leading to the formation of increasingly complex structures such as protofibrils that ultimately form insoluble (mature) fibrils. Oligomers are deemed particularly toxic because they serve as seeds that propagate the process. These seeds can also perforate neuronal cell membranes and disrupt homeostasis.^16,25,26 While studies have shown varying results, the consensus has been that fibrils are non-toxic or at least less toxic. In contrast, oligomers are now regarded as pathological. Recent research has indicated that fibril-associated proteins significantly influence amyloid formation and Alzheimer's disease pathology. Moreover, soluble oligomeric species of different proteins might be more toxic than larger fibrillar forms, challenging the correlation between pathological inclusions and disease.

We have explored the potential of soluble, surface-functionalized LAdN-CQDs to intervene in a PD-like phenotypic model. Our results reveal that LAdN-CQDs can disrupt the conversion of a model amyloid-forming protein (Fig. 1), interfering with its trajectory toward fibril formation and can also reduce levels of reactive oxygen species (ROS).

Fig. 1 Typical amyloidogenic trajectory showing the conversion of lysozyme into fibrils and the putative role of LAdN-CQDs on this mechanism.

Our results demonstrated that LAdN-CQDs effectively prevented the transformation of soluble monomers into mature fibrils of hen egg white lysozyme and neutralized reactive oxygen species when exposed to a variety of free radicals and hydroxyl generators. Furthermore, LAdN-CQDs were preincubated with the neuroblastoma-derived SHSY-5Y cell line to evaluate their protective effect against cell death caused by these stressors, demonstrating no cytotoxicity at concentrations up to 5 mg mL⁻¹ in comparison to the controls.

As previously mentioned, since each aberrant process affects the other, the multifaceted interventional approach of LAdN-CQDs is more impactful than the individual contributions. The findings indicate the development of a relatively new form of sustainable carbon nanomaterial, viz. LAdN-CQDs, for addressing amyloid-associated disorders.^27–29

Materials

All materials and reagents were obtained commercially. Lactic acid (Sigma-Aldrich); dimethyl sulfoxide (DMSO) (Fisher Chemical); ethylenediamine (Fisher Scientific, USA); Annexin V-FITC apoptosis kit (Abnova, USA); carboxy-H2DCFDA (Invitrogen, USA); quinine sulfate fluorescence (AnaSpec, USA); DPPH (Millipore-Sigma, USA); potassium persulfate (Fisher Chemical, UK); hen-egg white lysozyme (MP Biomedicals LLC, France); thioflavin T and rotenone (Sigma-Aldrich, USA); fetal bovine serum (FBS) (Atlanta Biologicals, USA); Dulbecco's modified Eagle's medium (DMEM) (Corning, USA); and SH-SY5Y cells (ATCC, USA). All reagents were of analytical grade.

Methods

LAdN-CQDs were synthesized via the hydrothermal method following the reaction of ethylenediamine with lactic acid in a nitrogen atmosphere.

A mixture of ethylene diamine and lactic acid was introduced with vigorous stirring in water. After 30 minutes of reaction, the mixture was kept at 0 °C, followed by the dropwise addition of a precooled aqueous solution of ammonium persulfate (4 mL, 0.5 M). The reaction was stirred overnight to ensure complete polymerization. The product was then washed with deionized water and dried under vacuum at 150 °C for 12 hours. The dried sample was transferred to a crucible and calcined at different temperatures (900 °C) for 10 hours under an N₂ atmosphere. After cooling, the product was collected and stored under vacuum. The carboxylic acid group modification on the surface of mesoporous carbon spheres was achieved by air calcination of the product at 300 °C for 1 hour. Following thermal treatment, the particles were dispersed in an aqueous medium and used for further characterization.

X-Ray diffraction analysis was performed using a Bruker diffractometer equipped with a Cu Kα radiation source. For the HRTEM analysis, the samples were drop-cast onto carbon-coated copper grids and examined using a HITACHI H9500 HRTEM with a LaB6 electron gun, operating at 300 kV with a resolution of 0.1 nm.

The fluorescence quantum yield (QY) is calculated using the equation.

F: integrated fluorescence intensity; OD: absorbance at 360 nm; A and S: arrest in and standard, respectively. The QY of the standard quinine sulfate is 0.55. The relative QY of the LAdN-CQDs was estimated at 0.53.¹⁶

Apoptosis assay

The potential of LAdN-CQDs to induce apoptosis was assessed by evaluating phosphatidylserine externalization in SH-SY5Y cells.^1,30,31 Initially, 100 000 cells were seeded using 1 mL of culture medium onto 24-well plates and incubated overnight at 37 °C in a 5% CO₂ atmosphere. The treatments were administered the next day, as follows. LAdN-CQDs at 2.5 and 5 mg mL⁻¹, 1% v/v PBS (vehicle), and 1 mM H₂O₂ (positive control for cell death). After 24 hours of incubation cells were prepared for the Annexin-V-FITC/PI assay kit (Beckman Coulter, Miami, FL, USA) following the manufacturer's instructions. Cells were centrifuged at 262 × g for 5 minutes, the supernatants removed, and then resuspended in 500 μL of PBS. This was followed by another centrifugation at 262 × g for 5 minutes, after which the pellets were resuspended in 100 μL of a mixture containing ice-cold 1× binding buffer, Annexin V-FITC, and propidium iodide (PI). The tubes were vortexed and incubated on ice in the dark for 30 minutes. Post-incubation, 300 μL of ice-cold 1× binding buffer was added to each tube, and immediate readings were taken using a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Data analysis was conducted using Kaluza 1.3 software (Beckman Coulter), with approximately 10 000 events per cell acquired per sample. To determine if LAdN-CQDs cause mitochondrial damage in SH-SY5Y cells, the MitoProbe JC-1 assay kit (Molecular Probes, Eugene, OR, USA; M34152) was employed, along with flow cytometry.^32,33 Cells were seeded at a density of 100 000 cells per mL of culture medium in 24-well plates and incubated overnight under optimal conditions (37 °C, 5% CO₂ atmosphere). The following day, the cells were treated with LAdN-CQDs at concentrations of 2.5 and 5 mg mL⁻¹, 1% v/v PBS (vehicle control), and 1 mM H₂O₂ (oxidative stress inducer), and incubated for 5 hours at 37 °C in a 5% CO₂ atmosphere. Subsequently, the cells were collected in flow cytometry tubes, centrifuged at 262 × g for 5 minutes, and the supernatants were discarded. The cells were then resuspended in 500 μL of PBS. Next, 5 μL of JC-1 fluorescent dye (2 μM final concentration) was added to each tube, and the samples were incubated at 37 °C (5% CO₂ atmosphere) for 30 minutes. The cells were washed by adding 1 mL of pre-warmed PBS to each tube, followed by centrifugation at 262 × g for 5 minutes. The cell pellets were resuspended in 500 μL of PBS and immediately analyzed using a Gallios flow cytometer. The FL1 and FL2 detectors were used to identify Alexa Fluor 488 and R-phycoerythrin in the samples, respectively. In healthy cells, JC-1 dye aggregates in the mitochondria, emitting a red signal due to the high membrane potential. When the mitochondrial membrane potential dissipates, JC-1 dye forms monomers and emits a green signal. For each sample, approximately 10 000 events were recorded, and data analysis was conducted using Kaluza 1.3 software.

Fibril detection assay

HEWL fibrils were produced by introducing approximately 1–3 mg mL⁻¹ of monomeric HEWL into a partially unfolding buffer (20 mM KH₂PO₄, 2 M guanidine hydrochloride, at 60 °C and 500 rpm for 4 hours in a Multi-therm Benchmark incubator shaker). In the treatment samples, various concentrations of LAdN-CQDs were added before incubation. The presence of fibrils was assessed by placing samples in a Quartz cuvette and adding 20 μM ThT. Fluorescence intensity was then measured with a DM45 Olis spectrofluorometer. Each experiment was conducted three times.

Circular dichroism measurement

For the secondary structural measurements dependent on LAdN-CQDs (0–0.5 mg mL⁻¹), CD spectra were collected at 25 °C with a bandwidth of 1 nm, a step interval of 1 nm, and a scan rate of 50 nm min⁻¹ (slit width of 0.02 mm; model JASCO J-1500, USA). Measurements were obtained using a quartz cell with a path length of 0.1 mm (190–260 nm). Various concentrations of LAdN-CQDs (0–5 mg mL⁻¹) were combined with 1 mg mL⁻¹ HEWL in a 5 mM Tris–HCl buffer. The LAdN-CQDs were introduced into the protein solution and incubated for 30 minutes before data collection. Scans were performed in triplicate, averaged, and the control buffer scans were subtracted from the sample spectra.

ROS assay

A DPPH solution (0.2 mg mL⁻¹) was prepared in 200 proof ethanol and stored at 4 °C. Ascorbic acid was used as the positive control. Various concentrations of LAdN-CQDs (10–100 μg mL⁻¹, prepared as a stock solution in ethanol) were added to individual vials containing DPPH. The vials were kept in the dark for 30 minutes to allow the reaction to occur before measuring the UV/Vis absorbance at 517 nm.

The percentage scavenging activity was calculated as follows:

Results and discussion

This study assesses the ability of LAdN-CQDs to modify amyloid-forming pathways and maintain intracellular equilibrium under oxidative stress.

Fig. 2(a) shows the reaction process for the formation of LAdN-CQDs. X-Ray diffractions (XRD) of the LAdN-CQDs clearly show the formation of CQDs by displaying 26.5°, which is related to the 002 plane of the nanomaterial, as shown in Fig. 2(b). HRTEM results (Fig. 2(c)) further confirm the uniform distribution of the nanoparticles with sizes ranging between 10–15 nm. Carbon XPS survey confirms the presence of the –C–N at 285.6 eV. The excitation-dependent fluorescence spectra are also displayed (Fig. 2(e)). The spectra show a shift in fluorescence emission towards longer wavelengths (red shift) correlated with the excitation wavelength. The excitation dependent emission reflects the presence of intra- and intermolecular differences in the LAdN-CQDs due to differences in the degree of functionalization.¹⁶ Nevertheless, strong luminescence was evident (Fig. 2(e)).

Fig. 2 (a) Schematic illustration of LAdN-CQD synthesis. (b) Powder X-ray diffraction pattern of LAdN-CQDs. (c) High-resolution transmission electron microscopy image of the synthesized LAdN-CQDs. (d) C-XPS survey of LAdN-CQDs. (e) Excitation-dependent fluorescence emission spectra of LAdN-CQDs.

The mitochondrial membrane potential (ΔΨ_m) arises from redox reactions linked to the Krebs cycle and acts as an intermediate energy storage utilized by ATP synthase to produce ATP.^1,30,31 Therefore, we investigated the mitochondrial transmembrane potential (ΔΨ_m) in neuroblastoma SH-SY5Y cells treated with LAdN-CQDs. Healthy cells preserve a mitochondrial inner membrane electrical potential, which is crucial to preventing apoptosis. SH-SY5Y cells underwent treatment with LAdN-CQDs, PBS, and H₂O₂, with the latter two serving as vehicle and positive control, respectively. Subsequent staining with JC-1 polychromatic dye and flow cytometry analysis was performed (Fig. 3(a)–(f)). The dye targets mitochondria and forms red fluorescent aggregates in cells with intact mitochondria (top left subpopulation; Fig. 3). A diminution in the red/green fluorescence intensity ratio indicates a loss of mitochondrial potential (bottom right subpopulation).

Fig. 3 Mitochondrial health is conserved in SHSY5Y cells treated with LAdN-CQDs. (a)–(e) Flow cytometry density plots of SHSY5Y cells treated with (a) LAdN-CQDs (2.5 mg mL⁻¹), (b) LAdN-CQDs (5 mg mL⁻¹), (c) untreated, (d) PBS, and (e) H₂O₂ (1 mM). (f) Percentage of cells with depolarized mitochondria after 5 h of treatment are shown, each treatment represents an average of three replicates. Cells were stained with the JC-1 polychromatic dye and analyzed via flow cytometry. Cells with depolarized mitochondria are shown in the right polygon versus healthy cells shown ungated in the top left. Statistical significance of the treatment compared to the vehicle control is indicated with the asterisks (***) p < 0.001.

Fig. 3 reveals no significant mitochondrial depolarization on experimental treatments. The impact of LAdN-CQDs on SH-SY5Y mitochondrial membrane potential is shown in Fig. 3(a), (b), and (f). The mitochondrial membrane potential was assessed following the treatment of cells with 2.5 and 5 mg mL⁻¹ of LAdN-CQDs for 5 hours, using flow cytometry for analysis. Controls included (d) vehicle (PBS), (c) untreated, and (e) H₂O₂. As expected, cells exposed to H₂O₂ displayed the highest percentage of depolarization (p < 0.001; Fig. 3(e) and (f)). Therefore, our results demonstrate that LAdN-CQDs do not perturb mitochondrial health in SH-SY5Y cells and will most likely not induce apoptosis.

To verify that LAdN-CQDs do not trigger apoptosis, phosphatidylserine externalization was assessed in SH-SY5Y cells treated with LAdN-CQDs (24 h; 2.5 and 5 mg mL⁻¹), PBS (vehicle), and H₂O₂ (apoptosis inducer) using the Annexin-V-FITC/PI assay. Cells treated with LAdN-CQDs showed no significant apoptosis (2.5 mg mL⁻¹ = 5.27% and 5 mg mL⁻¹ = 4.69%) compared to the vehicle control (PBS; 3.7%, Fig. 4). The data indicate that LAdN-CQDs do not cause apoptosis at the tested concentrations and are not cytotoxic to the extent investigated. Consequently, LAdN-CQDs are considered safe for use in human cells at concentrations ≤5 mg mL⁻¹, as depicted in Fig. 4(a), (b) and (f). The (d) vehicle (PBS), (c) untreated, and (e) hydrogen peroxide (+ve control) groups are displayed. No notable apoptosis was detected in cells treated with LAdN-CQDs relative to the vehicle control. The asterisk denotes the statistical significance of the data (***p < 0.001).

Fig. 4 LAdN-CQDs do not induce apoptosis in SHSY5Y cells. (a)–(e) Flow cytometry density plots used to quantify apoptosis and necrosis values. After 24 h of exposure to (a) LAdN-CQDs (2.5 mg mL⁻¹), (b) LAdN-CQDs (5 mg mL⁻¹), (c) untreated, (d) PBS, and (e) H₂O₂ (1 mM) induction of apoptosis was assessed using the Annexin-V PI FICT kit and flow cytometry. (f) Total apoptosis values of data in histograms (a=e). The following controls were included; untreated, PBS (vehicle) and H₂O₂ (positive control for cell death). Readings were taken with the Gallios flow cytometer and analyzed via the Kaluza software. Early (FITC+, PI−) and late (FITC+, PI+) apoptotic cells are denoted in the bottom and top right quadrants, respectively. Necrotic cells are represented in the top left quadrant (PI+). Statistical significance of the treatment against the vehicle control is denoted with asterisks (***) p < 0.001.

Fig. 5(a) illustrates the ThT fluorescence intensity trajectories that demonstrate the binding of ThT to mature HEWL fibrils and the effect of LAdN-CQDs on HEWL fibril formation. In the control experiment, represented in magenta, the introduction of ThT to mature fibrils showed a pronounced increase in fluorescence intensity, reaching saturation rapidly at a concentration of 5 mg mL⁻¹. Conversely, preincubating HEWL monomers with LAdN-CQDs (up to 5 mg mL⁻¹) resulted in the suppression of ThT fluorescence growth curves in a manner dependent on LAdN-CQD concentration. This dose-dependent decrease in ThT fluorescence indicates the inhibitory processes affecting the transformation of HEWL into mature fibrils. Under normal physiological conditions, the covalently bonded carbon core of the LAdN-CQDs does not dissociate into toxic entities. Additionally, LAdN-CQDs are highly biocompatible, showing comparable results to the vehicle control even at elevated doses, as evidenced by cytotoxicity assays. Generally, positively charged atoms are considered the most cytotoxic. However, our research indicates no toxicity for LAdN-CQDs, which are primarily functionalized with oxygen-containing groups on their surfaces. The discussion has been updated to reflect these findings.³⁴

Fig. 5 HEWL fibril formation assay: (a) ThT fluorescence intensity dependent on LAdN-CQD dosage; (b) CD spectra of fibrils correlating with LAdN-CQD dosage; (c) radical scavenging capability of LAdN-CQDs measured by the DPPH assay, varying with dosage.

To highlight the processes through which LAdN-CQDs disrupt the soluble monomer to mature fibril formation, we investigated the LAdN-CQD-dependent secondary structure of HEWL using circular dichroism (CD). The CD results suggest a LAdN-CQDs concentration-dependent reduction in HEWL's β-sheet structure. With an increasing N-CQDs concentration (up to 5 mg mL⁻¹), a total loss of HEWL β-sheet content was noted (Fig. 5(b)), signifying the prevention of mature fibril formation through the disruption of β-sheets by LAdN-CQDs.

The initiation and progression of neurodegeneration are linked to oxidative stress (increased ROS). This process represents a secondary pathway that triggers neuronal injury and subsequent cell death. Our investigation focused on whether LAdN-CQDs could act to reduce ROS levels that are associated with the onset of neurodegenerative diseases. For this purpose, we utilized the DPPH assay to evaluate the ROS scavenging ability of LAdN-CQDs. The analysis of the data (Fig. 5(c)) distinctly demonstrates the dose-dependent capability of LAdN-CQDs to counteract ROS levels. The results obtained from these quantum dots were comparable to the positive control (ascorbic acid), which displayed 80–90% DPPH scavenging activity at a similar concentration range.³⁵ Hydrogen atom donation from functional groups on the quantum dot surface to the DPPH radical effectively reduces the radical species, detected by a color change in the solution due to the loss of purple DPPH radical color. This process is typically attributed to the presence of carboxyl, hydroxyl and amino groups on the quantum dots. LAdN-CQDs are superior antioxidants when compared to other quantum dots previously documented. This comparison is emphasized in Table S1 (ESI†).

Conclusion

In this study, we synthesized LAdN-CQDs through a hydrothermal process involving ethylenediamine and lactic acid. The LAdN-CQDs demonstrated the ability to inhibit the transformation of HEWL into fibrillar aggregates. Additionally, the LAdN-CQDs exhibited free radical scavenging capabilities, as determined by a standard antioxidant activity assay. Notably, the LAdN-CQDs proved to be biocompatible at concentrations up to 5 mg mL⁻¹. Collectively, these findings highlight the potential of biocompatible LAdN-CQDs to independently intervene in the progression towards amyloidogenesis. The LAdN-CQDs have shown promise in preventing the harmful conversion of amyloid proteins, restoring cellular homeostasis by eliminating reactive oxygen species (ROS), and protecting cells from apoptosis and necrosis caused by mitochondrial toxicity. These characteristics suggest that LAdN-CQDs are a promising candidate for further experimental trials.

Author contributions

SE synthesized the LAdN-CQDs, characterized them and performed assays with the help of DAG, SV, JK, and MFS. RA, HS, SC and MN analyzed the data. MN conceived the topic, helped interpret the results and wrote the manuscript.

Data availability

All data will be made available upon request.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Mahesh Narayan acknowledges support from NIH 5R16GM145575-02 for this work. The work also required facilities of the Border Biomedical Research Center at The University of Texas at El Paso (UTEP). We are also thankful to all the staff members of the cellular Characterization and Biorepository Core facility, Border Biomedical Research Center, the University of Texas at El Paso (UTEP) supported by Grant U54MD007592.

References

1. J. Ahlawat and M. Narayan, Multifunctional carbon quantum dots prevent soluble-to-toxic transformation of amyloid and oxidative stress, ACS Sustainable Chem. Eng., 2022, 10(14), 4610–4622.
2. O. Dumbhare and S. S. Gaurkar, A Review of Genetic and Gene Therapy for Parkinson’s Disease, Cureus J. Med. Sci., 2023, 15(2), e34657.
3. M. R. Sahu, L. Rani, A. S. Kharat and A. C. Mondal, Could Vitamins Have a Positive Impact on the Treatment of Parkinson's Disease?, Brain Sci., 2023, 13(2), 272.
4. C. H. Schein, Distinguishing Curable from Progressive Dementias for Defining Cancer Care Options, Cancers, 2023, 15(4), 1055.
5. A. P. F. Waltrick, A. C. F. da Silva, B. A. de Mattos, Y. C. Chaves, R. M. W. de Oliveira, J. Prickaerts and J. M. Zanoveli, Long-term treatment with roflumilast improves learning of fear extinction memory and anxiety-like response in a type-1 diabetes mellitus animal model, Behav. Brain Res., 2023, 439, 114217.
6. Y. Hu, X. Wang, Y. Niu, K. He and M. Tang, Application of quantum dots in brain diseases and their neurotoxic mechanism, Nanoscale Adv., 2024, 6(15), 3733–3746.
7. D. Kim, J. M. Yoo, H. Hwang, J. Lee, S. H. Lee, S. P. Yun, M. J. Park, M. Lee, S. Choi and S. H. Kwon, Graphene quantum dots prevent α-synucleinopathy in Parkinson's disease, Nat. Nanotechnol., 2018, 13(9), 812–818.
8. Z. Saedi, M. J. Masroor, A. Jahangiri-Manesh, A. Rezaei, K. Siskova and M. Nikkhah, A Label-Free Sensor Array Based on Carbon Quantum Dots for Detection of α-Synuclein Oligomers in Saliva, Anal. Chem., 2024, 12246–12253.
9. J.-Y. Lee, A. Martin-Bastida, A. Murueta-Goyena, I. Gabilondo, N. Cuenca, P. Piccini and B. Jeon, Multimodal brain and retinal imaging of dopaminergic degeneration in Parkinson disease, Nat. Rev. Neurol., 2022, 18(4), 203–220.
10. D. Giraldo-Berrio, M. Mendivil-Perez, C. Velez-Pardo and M. Jimenez-Del-Rio, Rotenone Induces a Neuropathological Phenotype in Cholinergic-like Neurons Resembling Parkinson's Disease Dementia (PDD), Neurotoxic. Res., 2024, 42(3), 1–24.
11. R. K. Das, S. Panda, C. S. Bhol, S. K. Bhutia and S. Mohapatra, N-doped carbon quantum dot (NCQD)-Deposited carbon capsules for synergistic fluorescence imaging and photothermal therapy of oral cancer, Langmuir, 2019, 35(47), 15320–15329.
12. S. A. Shaik, S. Sengupta, R. S. Varma, M. B. Gawande and A. Goswami, Syntheses of N-doped carbon quantum dots (NCQDs) from bioderived precursors: a timely update, ACS Sustainable Chem. Eng., 2020, 9(1), 3–49.
13. V. Singh, S. Kashyap, U. Yadav, A. Srivastava, A. V. Singh, R. K. Singh, S. K. Singh and P. S. Saxena, Nitrogen doped carbon quantum dots demonstrate no toxicity under in vitro conditions in a cervical cell line and in vivo in Swiss albino mice, Toxicol. Res., 2019, 8(3), 395–406.
14. S. Zhang, X. Pei, Y. Xue, J. Xiong and J. Wang, Bio-safety assessment of carbon quantum dots, N-doped and folic acid modified carbon quantum dots: A systemic comparison, Chin. Chem. Lett., 2020, 31(6), 1654–1659.
15. C. Zhao, X. Wang, L. Wu, W. Wu, Y. Zheng, L. Lin, S. Weng and X. Lin, Nitrogen-doped carbon quantum dots as an antimicrobial agent against Staphylococcus for the treatment of infected wounds, Colloids Surf., B, 2019, 179, 17–27.
16. E. D. Guerrero, A. M. Lopez-Velazquez, J. Ahlawat and M. Narayan, Carbon Quantum Dots for Treatment of Amyloid Disorders, ACS Appl. Nano Mater., 2021, 4(3), 2423–2433,  DOI:10.1021/acsanm.0c02792.
17. M. Abd Elkodous, H. A. Hamad, M. I. A. A. Maksoud, G. A. M. Ali, M. El Abboubi, A. G. Bedir, A. A. Eldeeb, A. A. Ayed, Z. Gargar and F. S. Zaki, Cutting-edge development in waste-recycled nanomaterials for energy storage and conversion applications, Nanotechnol. Rev., 2022, 11(1), 2215–2294.
18. Y. Yang, Q. Wang, G. Li, W. Guo, Z. Yang, H. Liu and X. Deng, Cysteine-Derived Chiral Carbon Quantum Dots: A Fibrinolytic Activity Regulator for Plasmin to Target the Human Islet Amyloid Polypeptide for Type 2 Diabetes Mellitus, ACS Appl. Mater. Interfaces, 2023, 2617–2629.
19. R. K. Mani, K. Chandan, D. R. Bharathi and S. S. Ahmed, Quantum dots and its advancement in the treatment of Parkinson's disease: a review, J. Innovations Appl. Pharm. Sci., 2022, 61–63.
20. M. P. T. Prabhu and N. Sarkar, Inhibitory effects of carbon quantum dots towards hen egg white lysozyme amyloidogenesis through formation of a stable protein complex, Biophys. Chem., 2022, 280, 106714.
21. C.-L. Li, C.-M. Ou, C.-C. Huang, W.-C. Wu, Y.-P. Chen, T.-E. Lin, L.-C. Ho, C.-W. Wang, C.-C. Shih and H.-C. Zhou, Carbon dots prepared from ginger exhibiting efficient inhibition of human hepatocellular carcinoma cells, J. Mater. Chem. B, 2014, 2(28), 4564–4571.
22. A. Sachdev and P. Gopinath, Green synthesis of multifunctional carbon dots from coriander leaves and their potential application as antioxidants, sensors and bioimaging agents, Analyst, 2015, 140(12), 4260–4269,  10.1039/C5AN00454C.
23. K. M. Tripathi, A. K. Sonker, S. K. Sonkar and S. Sarkar, Pollutant soot of diesel engine exhaust transformed to carbon dots for multicoloured imaging of E. coli and sensing cholesterol, RSC Adv., 2014, 4(57), 30100–30107,  10.1039/C4RA03720K.
24. F. Di Domenico, E. Head, D. A. Butterfield and M. Perluigi, Oxidative stress and proteostasis network: culprit and casualty of Alzheimer’s-like neurodegeneration, Adv. Geriatr., 2014, 2014, 527518.
25. M. G. Iadanza, M. P. Jackson, E. W. Hewitt, N. A. Ranson and S. E. Radford, A new era for understanding amyloid structures and disease, Nat. Rev. Mol. Cell Biol., 2018, 19(12), 755–773,  DOI:10.1038/s41580-018-0060-8.
26. M. Dabur, J. A. Loureiro and M. C. Pereira, The current state of amyloidosis therapeutics and the potential role of fluorine in their treatment, Biochimie, 2022, 123–135.
27. E. Brandi, Role of Neuroinflammation in Parkinson's Disease and Related Disorders, Department of Experimental Medical Sciences, Faculty of Medicine, Lund…, PhD thesis, 2022.
28. R. Atchudan, S. Chandra Kishore, P. Gangadaran, T. N. Jebakumar Immanuel Edison, S. Perumal, R. L. Rajendran, M. Alagan, S. Al-Rashed, B.-C. Ahn and Y. R. Lee, Tunable fluorescent carbon dots from biowaste as fluorescence ink and imaging human normal and cancer cells, Environ. Res., 2022, 204, 112365,  DOI:10.1016/j.envres.2021.112365.
29. P. Krishnaiah, R. Atchudan, S. Perumal, E.-S. Salama, Y. R. Lee and B.-H. Jeon, Utilization of waste biomass of Poa pratensis for green synthesis of n-doped carbon dots and its application in detection of Mn²⁺ and Fe³⁺, Chemosphere, 2022, 286, 131764,  DOI:10.1016/j.chemosphere.2021.131764.
30. J. Ahlawat, G. Henriquez, A. Varela-Ramirez, R. Fairman and M. Narayan, Gelatin-derived carbon quantum dots mitigate herbicide-induced neurotoxic effects in vitro and in vivo, Biomater. Adv., 2022, 137, 212837.
31. G. Henriquez, J. Ahlawat, R. Fairman and M. Narayan, Citric Acid-Derived Carbon Quantum Dots Attenuate Paraquat-Induced Neuronal Compromise In Vitro and In Vivo, ACS Chem. Neurosci., 2022, 13(16), 2399–2409.
32. D. Nuzzo, M. Frinchi, C. Giardina, M. Scordino, M. Zuccarini, C. De Simone, M. Di Carlo, N. Belluardo, G. Mudò and V. Di Liberto, Neuroprotective and Antioxidant Role of Oxotremorine-M, a Non-selective Muscarinic Acetylcholine Receptors Agonist, in a Cellular Model of Alzheimer Disease, Cell. Mol. Neurobiol., 2022, 1–16.
33. K. Cuttler, D. de Swardt, L. Engelbrecht, J. Kriel, R. Cloete and S. Bardien, Neurexin 2 p. G849D variant, implicated in Parkinson's disease, increases reactive oxygen species, and reduces cell viability and mitochondrial membrane potential in SH-SY5Y cells, J. Neural Transm., 2022, 129(12), 1435–1446.
34. M. Havrdova, K. Hola, J. Skopalik, K. Tomankova, M. Petr, K. Cepe, K. Polakova, J. Tucek, A. B. Bourlinos and R. Zboril, Toxicity of carbon dots – Effect of surface functionalization on the cell viability, reactive oxygen species generation and cell cycle, Carbon, 2016, 99, 238–248,  DOI:10.1016/j.carbon.2015.12.027.
35. G. K. More and R. T. Makola, In-vitro analysis of free radical scavenging activities and suppression of LPS-induced ROS production in macrophage cells by Solanum sisymbriifolium extracts, Sci. Rep., 2020, 10(1), 6493,  DOI:10.1038/s41598-020-63491-w.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb02104e

---