A procyanidine derivative carbon dot and a preparation method and application thereof

Abstract

The application provides procyanidine-derived carbon dots and a preparation method and application thereof, belongs to the technical field of biological medicines, and aims to solve the problems of poor targeting, low brain penetration rate, poor biocompatibility and single function in the field of treatment of neurodegenerative diseases in the prior art. The derived carbon dots are prepared by using procyanidine as a carbon source through a hydrothermal method, the derived carbon dots are spherical nanoparticles, the average diameter is 1-2 nm, the height of the morphology is 5-15 nm, the lattice spacing is 0.30-0.38 nm, the surface contains carboxyl and phenolic hydroxyl functional groups, and the derived carbon dots have a pi-pi conjugated structure. The application realizes active, multi-target and systematic intervention on the fibrillation process of alpha-synuclein, and provides a new material platform and technical route for the treatment of neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a proanthocyanidin-derived carbon dot, its preparation method, and its application. Background Technology

[0002] Neurodegenerative diseases are a class of neurological disorders characterized by progressive neuronal loss and dysfunction. Among these, synucleinopathies such as Parkinson's disease and Lewy body dementia are characterized by abnormal α-synuclein fibrillation as a core pathological feature. The abnormal aggregation of α-synuclein forms insoluble amyloid fibers, which deposit within neurons, leading to cytotoxicity, oxidative stress, and mitochondrial dysfunction, ultimately driving neuronal death. Therefore, developing effective therapeutic strategies targeting this core pathological target of α-synuclein fibers is an important research direction in the treatment of neurodegenerative diseases.

[0003] Currently, intervention strategies targeting α-synuclein filaments mainly include the following categories: The first category is small molecule inhibitors, such as nilotinib. These compounds work by inhibiting the aggregation of α-synuclein or promoting its clearance. However, small molecule drugs generally suffer from poor targeting, making it difficult to specifically distinguish between pathological fibrillary α-synuclein and normal monomeric α-synuclein, easily interfering with normal physiological functions. In addition, small molecules have low brain penetration, making it difficult to achieve effective concentrations at the lesion site, and they also have potential systemic toxicity.

[0004] The second category is antibody therapy. Antibodies can specifically recognize and bind to α-synuclein, but antibody therapy is expensive, and due to the large molecular weight of antibodies, it is difficult for them to cross the blood-brain barrier and enter the central nervous system. More importantly, antibodies mainly act on extracellular proteins and have limited ability to clear existing intracellular fibrils, thus failing to effectively intervene in intracellular pathological processes.

[0005] The third category is strategies based on metal nanoparticles or polymer nanomaterials. Nanomaterials, utilizing their size effect and surface modifiability, can intervene in α-synuclein filaments to a certain extent. However, many synthetic nanomaterials face poor biocompatibility issues, and their long-term toxicity remains unclear, making it difficult to meet the safety requirements for long-term use in the central nervous system. Furthermore, existing nanomaterials have relatively limited functions, with most only exhibiting a single function of inhibiting aggregation or promoting clearance, making it difficult to simultaneously achieve the degradation of existing filaments and the inhibition of new filament formation.

[0006] The aforementioned shortcomings of existing technologies have remained unresolved for a long time, and the fundamental difficulties lie in the following three aspects: First, the aggregation mechanism of α-synuclein is complex, involving multi-stage conformational changes from soluble monomers to oligomers and then to mature fibers, requiring precise intervention in the pathological process while avoiding interference with its normal physiological function; Second, developing a delivery system that can efficiently penetrate the blood-brain barrier and specifically target diseased neurons is extremely challenging, requiring synergistic design that takes into account size effects, surface chemistry, and targeting capabilities; Third, the design of materials that combine highly efficient degradable fibers, low toxicity, and good biodegradability is a multidisciplinary problem, and traditional synthetic materials cannot simultaneously meet the above requirements.

[0007] Therefore, there is an urgent need to develop a multifunctional nanomaterial that can efficiently degrade existing α-synuclein fibers, inhibit the formation of new fibers, has good biocompatibility, low toxicity, and the ability to be delivered across the blood-brain barrier. Summary of the Invention

[0008] This application provides a proanthocyanidin-derived carbon dot, its preparation method, and its application, aiming to solve the problems of poor targeting, low brain penetration, poor biocompatibility, and limited function in the field of neurodegenerative disease treatment in the prior art.

[0009] To solve the above problems, the technical solution adopted in this application is as follows: In the first aspect, this application proposes a proanthocyanidin-derived carbon dot, which is prepared by hydrothermal method using proanthocyanidin as carbon source. The derived carbon dot is a spherical nanoparticle with an average diameter of 1-2 nm, a morphological height of 5-15 nm, and a lattice spacing of 0.30-0.38 nm. Its surface contains carboxyl and phenolic hydroxyl functional groups and has a π-π conjugated structure.

[0010] Furthermore, the derived carbon dots are composed of C and O elements, with a C content of 60-80% and an O content of 15-30%.

[0011] Furthermore, the surface of the derived carbon dots contains C=O bonds, CH bonds, and CO bonds.

[0012] Furthermore, the carboxyl and phenolic hydroxyl groups are retained from the functional groups derived from the proanthocyanidin precursor.

[0013] Furthermore, the derived carbon dots exhibit photoluminescence properties.

[0014] Furthermore, the surface of the derived carbon dots is modifiable.

[0015] Furthermore, the carbon dots contain carbon cores with graphitized structures.

[0016] Furthermore, the carbon dots are formed by the pyrolysis and carbonization of proanthocyanidins at 180-250℃, and the functional groups of the proanthocyanidin precursor are retained on the surface of the carbon dots after pyrolysis.

[0017] Secondly, this application proposes a method for preparing proanthocyanidin-derived carbon dots as described in the first aspect, comprising: Step 1: dissolving proanthocyanidins in a mixed solvent of anhydrous ethanol and ultrapure water, and dispersing by ultrasonication; Step 2: transferring the ultrasonically dispersed solution to a high-pressure reactor and heating at 180-250℃ for 6-16 hours; Step 3: cooling the reaction solution, filtering, collecting the filtrate, removing insoluble residues; dialysis purification, lyophilizing, and storing at low temperature.

[0018] Further, in step 1, the ratio of proanthocyanidins to the mixed solvent is 1:50-200, and the volume ratio of anhydrous ethanol to ultrapure water is 1:2-4.

[0019] Furthermore, the ultrasonic dispersion time is 15-60 min.

[0020] Furthermore, in step 2, the high-pressure reactor is a high-pressure reactor lined with Teflon.

[0021] Furthermore, in step 3, the filtration uses a 0.10-0.45μm microfiltration membrane.

[0022] Furthermore, the dialysis purification uses a dialysis membrane with a molecular weight cutoff of 500-2000 Da, the dialysis time is 4-16 hours, and the ultrapure water is replaced every 1-4 hours.

[0023] Furthermore, the low-temperature storage temperature is -80 to 4°C.

[0024] Use of the proanthocyanidin-derived carbon dots described in the first aspect or the proanthocyanidin-derived carbon dots prepared by the method described in the second aspect in the preparation of formulations for intervening in the α-synuclein fibrosis process.

[0025] Furthermore, the preparation is used to treat synucleinosis, which is Parkinson's disease or Lewy body dementia.

[0026] Furthermore, the formulation serves as a research tool for studying protein fibrosis mechanisms or screening inhibitors.

[0027] Furthermore, the proanthocyanidin-derived carbon dots are co-incubated with α-synuclein pre-fabricated filaments at a concentration of 0.5-2 mg / mL in PBS buffer at 35-40°C and 500-1500 rpm for 1-14 days; the pH of the PBS buffer is 7.0-7.6.

[0028] Furthermore, the proanthocyanidin-derived carbon dots were applied at a concentration of 0.01-0.05 mg / mL to an SH-SY5Y cell model induced by 10-50 μM α-synuclein pre-fibrils, and were administered as a pretreatment.

[0029] Furthermore, the formulation is also used for diagnostic imaging based on the photoluminescence properties of the carbon dots.

[0030] Furthermore, the formulation is delivered across the blood-brain barrier and targets intracellular α-synuclein filaments.

[0031] Furthermore, the transmembrane transport efficiency of the proanthocyanidin-derived carbon dots in the in vitro blood-brain barrier model reaches over 90% within 60-90 minutes; the formulation is also used to downregulate the pathological phosphorylation level of intracellular α-synuclein at the Ser129 site; the proanthocyanidin-derived carbon dots reduce the phosphorylation substrate recognized by kinases or change the protein conformation by degrading intracellular pathological α-synuclein aggregates, thereby reducing the pathological phosphorylation load.

[0032] A method for evaluating the intervention effect of proanthocyanidin-derived carbon dots on the pathological aggregation of α-synuclein in a cell model includes the following steps: Step 1: Establishing a pathological aggregation model by treating SH-SY5Y cells with α-synuclein prefabricated filaments; Step 2: Co-treating the cells with proanthocyanidin-derived carbon dots and α-synuclein prefabricated filaments; Step 3: Detecting the expression levels of α-synuclein monomers, oligomers, aggregates, and phosphorylated α-synuclein in the cells by Western blotting; Step 4: Detecting the distribution and fluorescence intensity of phosphorylated α-synuclein positive inclusion bodies in the cells by immunofluorescence staining; Step 5: Quantitatively analyzing the inhibitory effect of proanthocyanidin-derived carbon dots on the pathological aggregation and phosphorylation of α-synuclein.

[0033] A method for assessing the ability of proanthocyanidin-derived carbon dots to cross the blood-brain barrier includes the following steps: Step 1: Culture bEnd.3 cells in the upper chamber of a Transwell chamber to form a tight junction monolayer; Step 2: Seed SH-SY5Y cells in the lower chamber; Step 3: Add proanthocyanidin-derived carbon dots or proanthocyanidin monomers to the upper chamber and confirm the integrity of the monolayer by detecting transmembrane resistance; Step 4: Measure the transport efficiency of proanthocyanidin-derived carbon dots or proanthocyanidin monomers in the lower chamber at different time points; Step 5: Compare the differences in transmembrane transport between proanthocyanidin-derived carbon dots and proanthocyanidin monomers.

[0034] One or more technical solutions proposed in this application have at least the following technical effects: (i) Proanthocyanidins possess good biocompatibility and anti-inflammatory activity, but lack the ability to cross the blood-brain barrier and target protein fibers. Carbon dots exhibit nanoscale effects and surface modifiability, but traditional carbon dot surfaces lack chemical groups that specifically bind to proteins. This application structurally couples the two at the atomic / molecular level: after pyrolysis and carbonization, the aromatic skeleton of proanthocyanidins is reorganized into the graphitized carbon core of the carbon dot, endowing the carbon dot with photoluminescent properties; the carboxyl and phenolic hydroxyl groups at its edges are retained on the carbon dot surface, endowing the carbon dot with biorecognition activity. This core-shell structural synergy enables carbon dots to simultaneously possess the dual capabilities of reaching targets (nanoscale crossing of the blood-brain barrier) and recognizing targets (functional group-specific binding to fibers), both of which are indispensable; neither proanthocyanidins alone nor carbon dots alone can simultaneously meet the above requirements.

[0035] (ii) The carboxyl and phenolic hydroxyl groups on the surface of the carbon dots do not exist in isolation, but rather form a synergistic combination consistent with the typical biological functional groups of proanthocyanidin precursors. The carboxyl group carries a negative charge and can bind to the positively charged region on the surface of α-synuclein filaments through electrostatic interactions; the phenolic hydroxyl group can act as a hydrogen bond donor, forming hydrogen bonds with the peptide carbonyl groups in the β-sheet structure of the filament; simultaneously, the π-π conjugated structure of the carbon core can generate hydrophobic interactions with aromatic amino acid residues in the filament. The synergistic superposition of the above three forces (electrostatic, hydrogen bonding, and hydrophobic) makes the binding affinity of carbon dots to α-synuclein filaments significantly higher than that of a single interaction mode, thereby achieving specific targeting of pathological filaments. This multi-point recognition mechanism is something that proanthocyanidin monomers lack, as they lack the local concentration effect brought about by the lack of nanoscale size and the additional hydrophobic interactions brought about by the π-π conjugated structure of the carbon core.

[0036] (III) Most existing technologies are limited to inhibiting the abnormal aggregation of α-synuclein monomers, and are ineffective against already formed mature fibers. The carbon dots in this application specifically bind to the β-sheet structure of the fiber core through surface functional groups, disrupting fiber stability and inducing its dissociation into short fragments, thus achieving active degradation. Simultaneously, the carbon dots inhibit the conformational transition of monomers into fibers, blocking the formation of new fibers. The degradation of existing fibers and the inhibition of new fiber formation are not independent functions, but rather synergistic over time: the removal of deposited pathological fibers creates conditions for cell function recovery, while the inhibition of new fiber formation prevents a vicious cycle of pathological progression. This dual-functional synergy enables the carbon dots to exhibit therapeutic resilience not found in single-function drugs during intervention in fibrosis.

[0037] (iv) The neurotoxicity of α-synuclein fibers is not driven by a single mechanism, but rather amplified through a vicious cycle cascade: "fiber deposition → ROS generation → mitochondrial damage → further increase in ROS → neuronal death." The carbon dots in this application target multiple nodes in the aforementioned pathological chain simultaneously, forming a synergistic intervention network: upstream, the carbon dots degrade fibers and inhibit aggregation, eliminating the initial inducing factor for ROS generation; midstream, the carbon dots clear existing ROS through the antioxidant activity derived from proanthocyanidins, alleviating oxidative stress; downstream, the carbon dots protect the mitochondrial membrane potential, restoring the cell's energy metabolism function and disrupting the positive feedback loop between ROS and mitochondrial damage. The synergistic intervention of these three targets is not a simple additive effect, but rather generates an exponential protective gain by disrupting the vicious cycle. Clearing ROS alone without degrading fibers results in continued ROS generation induced by fibers; protecting mitochondria alone without clearing ROS results in continued ROS damage to mitochondria. Only by acting simultaneously on the upstream, midstream, and downstream can the pathway to neuronal death be fundamentally blocked.

[0038] (v) The photoluminescence properties of carbon dots originate from the synergistic luminescence mechanism of their internal graphitized carbon cores and surface defect states. This property is not possessed by proanthocyanidin monomers, but rather is a value-added effect of the carbon dot formation process. This fluorescence property enables carbon dots to function as imaging probes while simultaneously playing a role in fiber degradation and neuroprotection, allowing for real-time tracking of their distribution in vivo and the dynamic process of fiber degradation. The integration of therapeutic (fiber degradation) and diagnostic (fluorescence imaging) functions into a single nanoparticle avoids the dosage coordination problems and metabolic burden caused by the separate administration of therapeutic agents and contrast agents in the traditional separation of diagnosis and treatment, achieving true theranostics.

[0039] (vi) Effective Degradation of Intracellular Pathological Aggregates and Significant Downregulation of Pathological Phosphorylation: The PCDs provided in this application significantly degraded endogenous α-Syn pathological aggregates induced by exogenous α-Syn PFFs in the SH-SY5Y cell model. Western blot results showed that after PCD treatment, the high molecular weight SDS-resistant aggregation band (>180kDa) was significantly weakened, while the dimer (~35kDa) and trimer (~60kDa) signals were enhanced, indicating that PCDs could effectively dissociate the formed dense aggregates and restore the soluble oligomeric state of α-Syn. More importantly, PCDs significantly reduced the pathological phosphorylation level of α-Syn at the Ser129 site. In the cell model induced by α-Syn PFFs, the expression level of p-α-Syn was significantly increased, while after co-treatment with PCDs, the p-α-Syn level decreased to near the normal control group. Immunofluorescence staining further confirmed that the number and fluorescence intensity of p-α-Syn positive inclusion bodies in cells were significantly reduced after PCD treatment. This effect suggests that PCDs not only clear aggregates through physical action, but may also reduce pathological phosphorylation modifications upstream by altering protein conformation or blocking kinase recognition of substrates, thus achieving a deeper level of intervention in the pathological process of α-synuclein.

[0040] (vii) Highly efficient blood-brain barrier penetration: Validated using an in vitro Transwell BBB model, the PCDs of this application achieved a transmembrane transport efficiency of over 90% within 60-90 minutes, while the transport efficiency of proanthocyanidin monomers (PC) was less than 20%. The nano-size effect and surface functional group characteristics of PCDs endow them with excellent BBB penetration ability, solving the problem of low delivery efficiency of natural polyphenolic compounds to the central nervous system, and providing direct evidence for carbon dots to reach the lesion site and exert therapeutic effects in vivo.

[0041] In summary, this application, through the selection of proanthocyanidin carbon sources, the design of a controllable hydrothermal carbonization process, and the retention of multifunctional surface groups, creates a functionally inherited carbon dot with intrinsic synergy across four levels: material structure, surface chemistry, functional output, and pathological intervention. This carbon dot is not a simple mixture of proanthocyanidins and carbon dots, but rather a unified entity reorganized at the nanoscale with emerging properties. It is this deep-seated synergy that enables the carbon dot to overcome the functional limitations of existing technologies that only inhibit aggregation or promote clearance, achieving active, multi-target, and systematic intervention in the α-synuclein fibrosis process, thus providing a novel material platform and technological approach for the treatment of neurodegenerative diseases. Attached Figure Description

[0042] Figure 1 This is a schematic diagram illustrating the mechanism by which PCDs degrade α-Syn PFFs within cells according to this application. Figure 2 This is a method for synthesizing proanthocyanidin carbon dots (PCDs); Figure 3 This is a schematic diagram illustrating the aggregation of α-Syn monomers and the degradation of α-Syn fibrous tissues by the PCDs of this application; Figure 4 Characterization and properties of PCDs in this application; Among them, (a) is the TEM image and particle size distribution statistics of PCDs; (b) shows the AFM two-dimensional topography and thickness distribution curve of the PCDs; (c) is the AFM 3D plot of PCDs; (d) is a high-resolution transmission electron microscope image of PCDs; (e) shows the UV-VIS absorption spectrum of PCDs; (f) Comparison of the solution colors of PC and PCDs under white light and ultraviolet light; (g) shows the fluorescence emission spectrum of PCDs; (h) is the Zeta potential diagram of PCDs; (i) shows the FT-IR spectra of PCDs; (j) shows the XPS full-spectrum scan results of PCDs; (k) represents the high-resolution XPS C1s and O1s energy spectra of PCDs; Figure 5 This application describes the effects of PCDs on α-Syn aggregation and fiber degradation. Among them, (a) is a schematic diagram of the aggregation (1 mg / mL) of α-Syn monomers and the degradation (1 mg / mL) of α-Syn PFFs in the presence or absence of PCDs (1 mg / mL); (b) The kinetics of α-Syn monomer aggregation are reflected by monitoring the intensity of ThT fluorescence; (c) The kinetics of α-Syn PFFs degradation are observed by monitoring the intensity of ThT fluorescence; (d) TEM images of the α-Syn aggregation process under conditions of no other substances (top) and PCDs (bottom); (e) TEM images of α-Syn PFFs at different time points (1, 3, 5, 7 days) under conditions of no other substances (top) and PCDs (bottom); Figure 6 This application demonstrates the impact of PCDs on the α-Syn secondary structure. Figure 7 The PCDs in this application antagonize the cytotoxicity induced by α-Syn PFFs; Among them, (a) represents the optimal intervention concentration for PCDs screened by the CCK8 method; (b) Observation of the effect of Calcein / PI detection on the effect of PCDs intervention on α-Syn PFFs on cell viability and cytotoxicity under fluorescence microscopy; (c) Statistical analysis of the effects of PCD intervention on cell viability and cytotoxicity by Calcein / PI assay; data are expressed as x±s (n=3), * P < 0.05, ** P < 0.01, *** P < 0.001, ns indicates no significant difference; Figure 8 To alleviate α-Syn PFFs-induced oxidative stress and mitochondrial damage in PCDs; Among them, (a) shows the effect of PCDs intervention on α-Syn PFFs on intracellular ROS under a fluorescence microscope; (b) Statistical analysis of the effect of PCDs intervention on α-Syn PFFs on intracellular ROS; (c) The effect of PCDs intervention on α-Syn PFFs on intracellular mitochondrial membrane potential was observed under a fluorescence microscope; (d) Statistical analysis of the effect of PCDs intervention on α-Syn PFFs on intracellular mitochondrial membrane potential; data are expressed as x±s (n=3), ** P < 0.01, *** P < 0.001, ns indicates no significant difference; Figure 9 The effect of PCDs on the levels of p-α-Syn and α-Syn (monomers, oligomers, aggregates) proteins in SH-SY5Y cells; Among them, (a) shows the expression of p-α-Syn and α-Syn (monomers, oligomers, and aggregates) in SH-SY5Y cells; (b) Relative expression levels of p-α-Syn and α-Syn (monomer, oligomer, aggregate) proteins in SH-SY5Y cells; (c) shows the immunofluorescence expression of p-α-Syn in SH-SY5Y cells; (d) Relative immunofluorescence expression level of p-α-Syn in SH-SY5Y cells. All data are expressed as mean ± SD, ns. No significant difference was found, **P < 0.01, ***P < 0.001. Figure 10 To assess the ability of PC-Cy5.5 and PCDs-Cy5.5 to cross the in vitro BBB; Among them, (a) is the bEnd.3 monolayer cell model; (b) TEER values ​​were measured at 0 min and 90 min after the addition of PC-Cy5.5 and PCDs-Cy5.5; (c) Efficiency of PC-Cy5.5 and PCDs-Cy5.5 in vitro across the BBB; all data are expressed as mean ± SD, ***P < 0.001. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] It should be noted that if the embodiments of the present invention involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0045] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied simultaneously. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0046] Neurodegenerative diseases are a class of neurological disorders characterized by progressive neuronal loss and dysfunction. Among these, synucleinopathies such as Parkinson's disease and Lewy body dementia are characterized by abnormal α-synuclein fibrillation as a core pathological feature. The abnormal aggregation of α-synuclein forms insoluble amyloid fibers, which deposit within neurons, leading to cytotoxicity, oxidative stress, and mitochondrial dysfunction, ultimately driving neuronal death. Therefore, developing effective therapeutic strategies targeting this core pathological target of α-synuclein fibers is an important research direction in the treatment of neurodegenerative diseases.

[0047] Currently, intervention strategies targeting α-synuclein filaments mainly fall into the following categories: The first category is small molecule inhibitors, such as nilotinib. These compounds work by inhibiting the aggregation of α-synuclein or promoting its clearance. However, small molecule drugs generally suffer from poor targeting, making it difficult to specifically distinguish between pathological fibrillary α-synuclein and normal monomeric α-synuclein, easily interfering with normal physiological functions. Furthermore, small molecules have low brain penetration, making it difficult to achieve effective concentrations at the lesion site, and they also pose potential systemic toxicity. The second category is antibody therapy. Antibodies can specifically recognize and bind to α-synuclein, but antibody therapy is expensive, and due to the large molecular weight of antibodies, it is difficult to cross the blood-brain barrier to enter the central nervous system. More importantly, antibodies mainly act on extracellular proteins, with limited ability to clear already formed intracellular fibrils, failing to effectively intervene in intracellular pathological processes. The third category is strategies based on metal nanoparticles or polymer nanomaterials. Nanomaterials, utilizing their size effect and surface modifiability, can achieve intervention on α-synuclein filaments to a certain extent. However, many synthetic nanomaterials suffer from poor biocompatibility, and their long-term toxicity remains unclear, making it difficult to meet the safety requirements for long-term use in the central nervous system. Furthermore, existing nanomaterials have relatively limited functions, with most only exhibiting the ability to inhibit aggregation or promote clearance, making it difficult to simultaneously degrade existing fibers and inhibit the formation of new fibers. These shortcomings of existing technologies have remained unresolved for a long time, fundamentally due to the following three aspects: First, the aggregation mechanism of α-synuclein is complex, involving multi-stage conformational changes from soluble monomers to oligomers and then to mature fibers, requiring precise intervention in the pathological process while avoiding interference with normal physiological functions; second, developing delivery systems that can efficiently penetrate the blood-brain barrier and specifically target diseased neurons is extremely challenging, requiring synergistic design that considers size effects, surface chemistry, and targeting capabilities; third, designing materials that combine efficient fiber degradation, low toxicity, and good biodegradability is a multidisciplinary challenge, and traditional synthetic materials cannot simultaneously meet these requirements.

[0048] This application breaks through the technical bias of using simple small molecules as carbon sources in traditional carbon dot preparation. It selects proanthocyanidins, a natural plant polyphenol with inherent biological activity, as the carbon source. Through a controllable hydrothermal carbonization process, the characteristic functional groups of proanthocyanidins are preserved rather than destroyed while forming carbon dot nanostructures. This creates a functionally inherited carbon dot that combines the nanodelivery and photophysical properties of carbon dots with the biocompatibility and pathological intervention activity of proanthocyanidins, thereby achieving active intervention in the α-synuclein fibrosis process and multi-target neuroprotection.

[0049] Traditional carbon dot preparation uses simple molecules such as citric acid and glucose as carbon sources, pursuing carbon core purity and luminescence efficiency. However, the resulting products lack active sites on the surface that specifically interact with biomolecules. This application takes the opposite approach, using structurally complex proanthocyanidins as a carbon source. Leveraging their abundant aromatic ring structure and multiple phenolic hydroxyl groups, a graphitized carbon core with a π-π conjugated structure is formed during hydrothermal carbonization. Simultaneously, key functional groups such as carboxyl and phenolic hydroxyl groups are retained on the carbon dot surface. This functional inheritance design transforms the carbon dots from inert nanocarriers into functionalized nanoparticles with intrinsic bioactivity.

[0050] The first aspect of this invention provides a proanthocyanidin-derived carbon dot, which is prepared by a hydrothermal method using proanthocyanidins as the carbon source. Choosing proanthocyanidins as the carbon source leverages their inherent biocompatibility, anti-inflammatory properties, and abundant functional groups. This allows the prepared carbon dot to retain the bioactive functional groups of proanthocyanidins while possessing the nanoscale effect and photophysical properties of carbon dots, thereby achieving targeted intervention on α-synuclein fibers.

[0051] The derived carbon dots are spherical nanoparticles with an average diameter of 1-2 nm and a height of 5-15 nm. This size range endows the carbon dots with the basic properties of nanomaterials while meeting the size requirements for delivery across the blood-brain barrier. The lattice spacing is 0.30-0.38 nm, which corresponds to the layered structure of graphitized carbon materials, indicating that an ordered carbon core structure has formed inside the carbon dots.

[0052] The derived carbon dots contain carboxyl and phenolic hydroxyl functional groups on their surface. These carboxyl and phenolic hydroxyl groups are derived from the retained functional groups of the proanthocyanidin precursor, representing a continuation of the typical biological functional groups of proanthocyanidins after pyrolysis and carbonization on the carbon dot surface. The presence of surface carboxyl and phenolic hydroxyl groups endows the carbon dots with the following functions: first, specific binding to α-synuclein filaments through electrostatic interactions and hydrogen bonds; second, providing modifiable sites on the carbon dot surface to facilitate the subsequent connection of targeting ligands; and third, maintaining the dispersion stability of the carbon dots in aqueous solution.

[0053] The derived carbon dots possess a π-π conjugated structure. This π-π conjugated structure is the structural basis for the photoluminescence properties of the carbon dots, enabling them to emit fluorescence and thus providing a signal source for diagnostic imaging. Simultaneously, the π-π conjugated structure can interact with aromatic amino acid residues in α-synuclein filaments, enhancing the carbon dots' targeted binding ability to the filaments.

[0054] In some embodiments, the derived carbon dots contain carbon (C) and oxygen (O), with a C content of 60-80% and an O content of 15-30%. This elemental composition indicates that the carbon dots are predominantly composed of a carbon skeleton while retaining a high proportion of oxygen-containing functional groups, distinguishing them from conventional carbon dots that are predominantly composed of pure carbon cores. The high oxygen content directly corresponds to the abundant carboxyl and phenolic hydroxyl groups on the surface, which is the chemical basis for the bioactivity of the carbon dots.

[0055] In some embodiments, the surface of the derived carbon dots contains C=O bonds, CH bonds, and CO bonds. The C=O bonds correspond to carboxyl functional groups, the CO bonds correspond to oxygen-containing functional groups such as phenolic hydroxyl groups and ether bonds, and the CH bonds correspond to the carbon skeleton and residual organic groups. The combination of these chemical bonds constitutes the chemical environment of the carbon dot surface, making it both hydrophilic and reactive.

[0056] In some embodiments, the derived carbon dots possess photoluminescence properties. These properties stem from the synergistic effect of the graphitized carbon cores within the carbon dots and the surface defect states, enabling the carbon dots to emit fluorescence upon excitation at a specific wavelength. This property allows the carbon dots to serve not only as therapeutic carriers but also as imaging probes, enabling integrated diagnostic and therapeutic applications.

[0057] In some embodiments, the surface of the derived carbon dots contains functional groups that can be chemically modified. These chemically modifiable functional groups include carboxyl and phenolic hydroxyl groups, which can be linked to targeting ligands (such as transferrin, lactoferrin, RGD peptides, etc.) through chemical reactions such as amidation and esterification, thereby achieving active targeted delivery to diseased neurons.

[0058] In some embodiments, the carbon dots contain graphitized carbon cores. These graphitized carbon cores are composed of sp... 2 The hybridized carbon atom structure is the structural source of the photophysical properties of carbon dots. The degree of graphitization of the carbon core affects the fluorescence quantum yield and photostability of carbon dots, while the rigid structure of the carbon core provides good physical stability for carbon dots.

[0059] In some embodiments, the carbon dots are formed by the pyrolysis and carbonization of proanthocyanidins at 180-250°C. This temperature range represents a suitable window for the dehydration and carbonization reactions of proanthocyanidins to form carbon nuclei. If the temperature is too low, carbonization is incomplete, failing to form an effective carbon nucleus structure; if the temperature is too high, the functional groups of proanthocyanidins decompose excessively, resulting in loss of biological activity. Within this temperature range, the functional groups of the proanthocyanidin precursor remain on the surface of the carbon dots after pyrolysis, achieving the preparation of "functionally inherited" carbon dots.

[0060] A second aspect of this invention provides a method for preparing proanthocyanidin-derived carbon dots, comprising the following steps: Step 1: Dissolve proanthocyanidins in a mixed solvent of anhydrous ethanol and ultrapure water, and then disperse by ultrasonication. Anhydrous ethanol acts as a co-solvent to promote the dissolution of proanthocyanidins, while ultrapure water provides the water molecules required for the hydrothermal reaction. Ultrasonic dispersion ensures that proanthocyanidin molecules are uniformly dispersed in the solvent, forming a homogeneous precursor solution, thus ensuring the uniformity of the subsequent hydrothermal reaction.

[0061] Step 2: Transfer the sonicated solution to a high-pressure reactor and heat at 180-250℃ for 6-16 hours. The high-pressure reactor provides a sealed, high-pressure environment, keeping water in a liquid state at a temperature above its normal boiling point, promoting the dehydration, polymerization, and carbonization of proanthocyanidin molecules. The specified temperature and time range ensures that proanthocyanidins are fully carbonized to form carbon nuclei, while avoiding over-carbonization that could lead to complete decomposition of functional groups.

[0062] Step 3: After cooling the reaction solution, filter it, collect the filtrate, and remove insoluble residues; dialysis purification, lyophilization, and cryogenic storage. Filtration removes large insoluble impurities and collects the filtrate containing carbon dots. Dialysis purification removes small molecule byproducts and unreacted raw materials, improving the purity of the carbon dots. Lyophilization removes moisture to obtain a solid product, facilitating storage and subsequent use. Cryogenic storage inhibits the chemical degradation and aggregation of carbon dots, maintaining their stability.

[0063] In some embodiments, in step 1, the volume ratio of proanthocyanidins to the mixed solvent is 1:50-200 (g / mL), and the volume ratio of anhydrous ethanol to ultrapure water is 1:2-4. This volume ratio ensures that the proanthocyanidins have a suitable concentration in the solvent, guaranteeing sufficient dissolution while avoiding excessive viscosity that could affect dispersion. The volume ratio adjusts the polarity and dielectric constant of the solvent, influencing the dissolution state of the proanthocyanidins and the progress of the hydrothermal reaction.

[0064] In some embodiments, the ultrasonic dispersion time in step 1 is 15-60 minutes. The ultrasonic time affects the dispersion uniformity of the precursor; if the time is too short, the dispersion will be insufficient, and if the time is too long, it may lead to local overheating and degradation of the raw material.

[0065] In some embodiments, in step 2, the high-pressure reactor is a Teflon-lined high-pressure reactor. The Teflon lining has excellent corrosion resistance and chemical inertness, avoiding catalytic interference from the metal reactor wall on the hydrothermal reaction, and ensuring product purity and batch consistency.

[0066] In some embodiments, step 3 involves filtration using a 0.10-0.45 μm microfiltration membrane. The pore size range of the microfiltration membrane effectively traps large particulate impurities and agglomerates while allowing carbon dots to pass through, achieving preliminary purification and particle size screening of the product.

[0067] In some embodiments, in step 3, the dialysis purification uses a dialysis membrane with a molecular weight cutoff of 500-2000 Da, and the dialysis time is 4-16 hours, with ultrapure water replaced every 1-4 hours. The molecular weight cutoff of the dialysis membrane ensures that carbon dots are retained inside the membrane while small molecule impurities permeate. The dialysis time and water change frequency affect the purification efficiency; too short a time or too infrequent water changes result in insufficient purification, while too long a time increases operating costs.

[0068] In some embodiments, in step 3, the cryogenic storage temperature is -80 to 4°C. This temperature range covers everything from ultra-low temperature freezing to conventional refrigeration, providing suitable storage conditions for carbon points to maintain their physicochemical stability and biological activity.

[0069] A third aspect of the present invention provides the use of the above-mentioned proanthocyanidin-derived carbon dots in the preparation of formulations for intervening in the α-synuclein fibrosis process.

[0070] Aberrant fibrosis of α-synuclein is a core pathological mechanism in synucleinogenic diseases such as Parkinson's disease and Lewy body dementia. The formulation described herein intervenes in the fibrosis process through the interaction between the proanthocyanidin-derived carbon dots and α-synuclein fibers. This intervention includes degrading existing α-synuclein fibers and inhibiting the abnormal aggregation of α-synuclein monomers, thereby blocking the progression of neurotoxicity.

[0071] In some embodiments, the formulation is used to treat synucleinosis, such as Parkinson's disease or Lewy body dementia. The carbon dots are delivered across the blood-brain barrier and target intracellular α-synuclein fibers, exerting a dual effect of fiber degradation and neuroprotection at the lesion site.

[0072] In some embodiments, the formulation serves as a research tool for studying protein fibrosis mechanisms or screening inhibitors. The carbon dots can be used as model compounds to study the kinetics and structural transition mechanisms of α-synuclein fibrosis, or as positive controls or screening platforms to evaluate the antifibrotic activity of other candidate compounds.

[0073] In some embodiments, the proanthocyanidin-derived carbon dots are co-incubated with 0.5-2 mg / mL of α-synuclein pre-fabricated cellulose in PBS buffer (pH 7.0-7.6) at 35-40°C and 500-1500 rpm for 1-14 days. This concentration range ensures sufficient interaction opportunities between the carbon dots and the cellulose. The PBS buffer maintains a physiological pH environment to ensure the stability of the protein and carbon dots. The temperature and rotation speed simulate the molecular motion under physiological conditions, promoting collisions and binding between the carbon dots and the cellulose. The incubation time covers the kinetics of cellulose degradation, allowing the carbon dots to fully exert their depolymerization effect.

[0074] In some embodiments, the proanthocyanidin-derived carbon dots are applied at a concentration of 0.01-0.05 mg / mL to an SH-SY5Y cell model induced by 10-50 μM α-synuclein prefibrils, and are administered before α-synuclein prefibrils induction. This concentration range provides protection at the cellular level while avoiding cytotoxicity. This pretreatment administration method allows the carbon dots to be distributed in the cellular environment before fibrils induction, establishing a protective barrier in advance and antagonizing fibrils-induced neurotoxicity by alleviating oxidative stress and protecting mitochondrial function.

[0075] In some embodiments, the formulation is also used for diagnostic imaging based on the photoluminescence properties of the carbon dots. The fluorescence properties of the carbon dots enable them to be detected and tracked in vitro or in vivo, reflecting the presence, distribution, and degradation of α-synuclein filaments through changes in fluorescence signals, thus enabling real-time monitoring of the treatment process.

[0076] In some embodiments, the formulation is also used to downregulate the pathological phosphorylation level of intracellular α-synuclein at the Ser129 site. The carbon dots reduce the pathological phosphorylation load by degrading intracellular pathological α-synuclein aggregates, decreasing phosphorylation substrates recognized by kinases, or altering protein conformation.

[0077] In some embodiments, the formulation is delivered across the blood-brain barrier and targets intracellular α-synuclein filaments. The transmembrane transport efficiency of the carbon dots in an in vitro blood-brain barrier model reaches over 90% within 60-90 minutes, significantly higher than the transport efficiency of proanthocyanidin monomers, thus solving the problem of low delivery efficiency of natural polyphenolic compounds to the central nervous system.

[0078] Example 1: Preparation of proanthocyanidin-derived carbon dots 0.6 g of proanthocyanidins was dissolved in 15 mL of anhydrous ethanol and 45 mL of ultrapure water, and sonicated at room temperature for 30 min. The solution was then transferred to a 100 mL Teflon-lined high-pressure reactor and heated at 200 °C for 10 h. After cooling, the solution was filtered through medium-speed filter paper, and the filtrate was collected. The filtrate was then filtered through a 0.22 μm filter membrane to remove insoluble residues. The resulting solution was transferred to a dialysis membrane with an MWCO of 1000 Da and dialyzed in ultrapure water for 8 h, with the ultrapure water changed every 2 h during the dialysis process. The dialyzed solution was lyophilized, and the product was stored at -20 °C to obtain proanthocyanidin-derived carbon dots (PCDs). Figure 2 As shown, the above-mentioned hydrothermal synthesis process dehydrates and carbonizes the proanthocyanidin precursor through high temperature and high pressure, forming spherical nanoparticles with retained proanthocyanidin characteristic functional groups.

[0079] like Figure 1As shown, the prepared PCDs can be delivered into neurons across the blood-brain barrier. Inside the cell, they interact with α-synuclein pre-formed filaments (α-Syn PFFs), inducing fiber dissociation through the specific binding of surface functional groups to the β-sheet structure of the fibers, thus achieving active degradation of the intracellular fibers. In the above examples, the ratio of proanthocyanidins to the mixed solvent was 1:100 (g / mL), the volume ratio of anhydrous ethanol to ultrapure water was 1:3, the ultrasonic dispersion time was 30 min, the reaction temperature was 200℃, the reaction time was 10 h, the high-pressure reactor was a Teflon-lined high-pressure reactor, filtration was performed using a 0.22 μm microfiltration membrane, dialysis purification was performed using a dialysis membrane with a molecular weight cutoff of 1000 Da, the dialysis time was 8 h, the ultrapure water was replaced every 2 h, and the cryogenic storage temperature was -20℃.

[0080] Example 2: Structural characterization of proanthocyanidin-derived carbon dots The PCDs prepared in Example 1 were systematically characterized, and the results are as follows: the PCDs are uniform spherical nanoparticles. Figure 4 As shown in Figure a, transmission electron microscopy (TEM) images and particle size distribution statistics show that the average diameter of PCDs is 1.08 ± 0.35 nm. Figure 4 b and Figure 4 As shown in Figure c, the morphological height of the PCDs is 8.54 ± 1.17 nm, and the AFM three-dimensional plot further confirms their spherical morphology. Figure 4 As shown in d, the lattice spacing of PCDs is 0.34 nm, which meets the standard for nanomaterials.

[0081] PCDs contain C and O elements, such as Figure 4 As shown in Figure j, the XPS full-spectrum scan results indicate a C content of 70.12% and an O content of 25.27%. Figure 4 As shown in k, the high-resolution XPS C1s spectrum shows splitting peaks at approximately 288 eV (C=O) and 286 eV (CO), which are attributed to the characteristic binding energies of carboxyl and phenolic hydroxyl groups. The high-resolution XPS O1s spectrum further confirms the presence of oxygen-containing functional groups.

[0082] PCDs surfaces contain C=O bonds, CH bonds, and CO bonds. For example... Figure 4 As shown in i, the FT-IR spectrum shows that PCDs are at 1282 cm⁻¹ -1 1437cm -1 1611cm -1 and 3231cm -1 A characteristic absorption peak appeared at 1282 cm⁻¹. -1 The absorption peak at 1437 cm⁻¹ is attributed to the C=O bond. -1 The absorption peak at 1611 cm⁻¹ is attributed to the CH bond. -1and 3231cm -1 The absorption peak is attributed to the -OH bending vibration. The above characteristic absorption peak is related to the typical biological functional groups of the PC precursor, confirming that the functional groups of the proanthocyanidin precursor are retained on the carbon dot surface after pyrolysis.

[0083] PCDs have a π-π conjugated structure. For example... Figure 4 As shown in Figure e, compared with PC, the UV-Vis spectrum of the prepared PCD nanoparticles showed a significant absorption peak (n-π transition) at 416 nm, confirming that PCDs have the typical spectral characteristics of carbon dots.

[0084] PCDs exhibit photoluminescence properties. For example... Figure 4 As shown in f, the solution containing nanoparticles appears light brown under ambient light, but light green under 365nm ultraviolet light excitation, indicating that PCDs have significant photoluminescence capabilities. Figure 4 As shown in g, fluorescence spectroscopy analysis revealed that PCDs exhibited maximum emission intensity at 392.2 nm when excited at a wavelength of 320 nm. The solution containing nanoparticles appeared light brown under ambient light and light green under 365 nm ultraviolet light excitation, indicating that PCDs possess significant photoluminescence capabilities.

[0085] PCDs surfaces contain functional groups that can be chemically modified. For example... Figure 4 As shown in h, the Zeta potential measurement result is negative, indicating that it retains abundant functional groups derived from PC, namely carboxyl groups (-COOH) and phenolic hydroxyl groups (-OH). These functional groups can serve as chemical modification sites to connect targeted ligands.

[0086] The above embodiments correspond to the proanthocyanidin-derived carbon dots described in claims 1-4 of this invention, wherein the average diameter is 1.08±0.35nm falling within the range of 1-2nm, the morphological height is 8.54±1.17nm falling within the range of 5-15nm, the lattice spacing is 0.34nm falling within the range of 0.30-0.38nm, the C content is 70.12% falling within the range of 60-80%, the O content is 25.27% falling within the range of 15-30%, the excitation wavelength is 320nm and the emission wavelength is 392.2nm, corresponding to photoluminescence characteristics, the carboxyl and phenolic hydroxyl groups are derived from proanthocyanidin precursors, and the carbon dots are formed by the pyrolysis and carbonization of proanthocyanidins at 200℃.

[0087] Example 3: Intervention of PCDs on α-synuclein fibrosis α-Syn monomer / pre-formed cellulose (1 mg / mL) was dissolved in PBS buffer (pH=7.4) and incubated at 37℃ and 1000 rpm for 1, 3, 5, and 7 days. Simultaneously, 1 mg / mL of α-Syn monomer / pre-formed cellulose was administered during incubation, and changes in α-Syn morphology, degree of fibrilation, fibril expression level, and the proportion of protein secondary structure components were observed. Figure 3 As shown, PCDs inhibit abnormal aggregation of monomers and promote depolymerization of already formed fibers by interacting with α-Syn monomers or preformed fibers.

[0088] Results from thioflavin T (ThT) fluorescence detection and transmission electron microscopy (TEM) showed that α-Syn monomers did not assemble into mature amyloid fibers in the presence of PCDs, indicating that PCDs primarily inhibited the fibrosis of α-Syn monomers. Furthermore, PCDs could induce the dissociation of α-Syn PFFs into short fragments. Figure 5 As shown in Figure a, the schematic diagram of α-Syn monomer aggregation (1 mg / mL) and α-Syn PFF degradation (1 mg / mL) in the presence and absence of PCDs (1 mg / mL) clearly illustrates the dual role of PCDs. Figure 5 As shown in b, the intensity of ThT fluorescence can reflect the kinetics of α-Syn monomer aggregation. The ThT fluorescence intensity is significantly reduced in the presence of PCDs, indicating that monomer fibrillation is inhibited. Figure 5 As shown in Figure c, the intensity of ThT fluorescence can be used to reflect the kinetics of α-SynPFFs degradation. The decrease in ThT fluorescence intensity after PCD treatment indicates fiber depolymerization. Figure 5 As shown in d, TEM images of the α-Syn aggregation process under conditions without other substances show the formation of mature fibers; under PCD conditions, α-Syn monomers do not assemble into mature amyloid fibers. Figure 5 As shown in e, under conditions without other substances, α-Syn PFFs maintained their fiber morphology at different time points (1, 3, 5, and 7 days); under PCD conditions, the fibers gradually dissociated into short segments.

[0089] Circular dichroism (CD) spectroscopy was used to determine the proportions of secondary structures, and the CDNN algorithm was employed to analyze these proportions. For example... Figure 6 As shown in Figure a, the CD spectra of the α-Syn monomer after 7 days of incubation with and without PCDs show significant changes in secondary structure. Figure 6 As shown in b, the secondary structure fraction ratios of α-Syn fibers decomposed by α-Syn fibers and PCDs were calculated using the CDNN algorithm. After co-incubation with PCDs, the β-sheet component decreased from 61.4% to 2.0%. Figure 6As shown in Figure c, the CD spectra of α-Syn PFFs after 7 days of incubation with and without PCDs show significant changes in secondary structure. Figure 6 As shown in Figure d, the ratio of protein secondary structure content in α-Syn PFFs degraded by α-Syn PFFs and PCDs was calculated using the CDNN algorithm. After co-incubation with PCDs, the β-sheet fraction decreased from 60.6 to 19.9. The β-sheet is the core structural basis for the transformation of α-Syn from soluble monomers to insoluble amyloid filaments. After 7 days of incubation, α-Syn monomers formed α-Syn filaments. After co-incubation with PCDs, the β-sheet fraction decreased from 61.4 to 2.0. After 7 days of incubation, α-Syn PFFs, after co-incubation with PCDs, showed a decrease in the β-sheet fraction from 60.6 to 19.9. These results indicate that PCDs significantly inhibited the fibrosis process of α-Syn PFFs by effectively disrupting the core β-sheet structure, thus achieving the degradation of existing filaments and the inhibition of monomer aggregation.

[0090] The above embodiments correspond to the uses described in claims 8-9 of the present invention, wherein the concentration of PCDs is 1 mg / mL falling within the range of 0.5-2 mg / mL, the concentration of α-Syn pre-fabricated fibers is 1 mg / mL falling within the range of 0.5-2 mg / mL, the pH of the PBS buffer is 7.4 falling within the range of 7.0-7.6, the incubation temperature is 37℃ falling within the range of 35-40℃, the rotation speed is 1000 rpm falling within the range of 500-1500 rpm, and the incubation time is 1-7 days falling within the range of 1-14 days.

[0091] Example 4: Neuroprotective effect of PCDs on Parkinson's disease PCDs were applied to an α-Syn PFFs-induced SH-SY5Y cell model. CCK8 results showed that the optimal protective concentration of PCDs in the α-SynPFFs-induced SH-SY5Y cell model was 0.02 mg / mL. Figure 7 As shown in Figure a, the optimal concentration of PCDs was screened using the CCK8 assay, and the results showed that 0.02 mg / mL was the optimal protective concentration. Calcein / PI assays of the effects of PCDs on α-SynPFFs on cell viability and cytotoxicity showed that 30 μM α-Syn PFFs significantly induced cell death, while PCD pretreatment significantly inhibited α-Syn PFF-induced cell damage. Quantitative analysis of the percentage of live and dead cells also confirmed these results. Figure 7As shown in b, the effect of PCDs intervention on α-Syn PFFs on cell viability and cytotoxicity was observed under a fluorescence microscope using Calcein / PI assay. 30 μM α-Syn PFFs significantly induced cell death, while PCD pretreatment significantly inhibited α-Syn PFFs-induced cell damage. Figure 7 As shown in c, the statistical analysis of the effects of Calcein / PI assay on the cell viability and cytotoxicity of PCDs-mediated intervention in α-Syn PFFs further quantitatively confirmed the above results.

[0092] Intracellular ROS expression was detected using the DCFH-DA probe. ROS levels were relatively low in healthy SH-SY5Y cells and cells pretreated with PCDs. ROS expression significantly increased after induction with α-Syn PFFs, while PCD pretreatment significantly inhibited α-Syn PFF-induced intracellular ROS production. These results indicate that PCDs antagonize the neurotoxicity of α-Syn PFFs by alleviating oxidative stress. Figure 8 As shown in Figure a, the effect of PCDs intervention on α-Syn PFFs on intracellular ROS was observed under a fluorescence microscope. ROS levels were relatively low in healthy SH-SY5Y cells and cells pretreated with PCDs. ROS expression significantly increased after α-Syn PFFs induction, while PCD pretreatment significantly inhibited ROS production. Figure 8 As shown in b, statistical analysis of the effect of PCDs intervention on α-Syn PFFs on intracellular ROS further quantitatively confirmed the above results.

[0093] Mitochondrial membrane potential was detected using the JC-1 fluorescent probe. Compared to the control group, there was no significant difference in the fluorescence intensity ratio of JC-1 polymer to JC-1 monomer in the PCDs group, indicating that PCDs did not affect mitochondrial membrane potential. The fluorescence intensity ratio of JC-1 polymer to JC-1 monomer in the α-Syn PFFs group was significantly reduced, indicating that α-Syn PFFs decreased mitochondrial membrane potential. However, compared to the α-Syn PFFs group, cells pretreated with PCDs and then induced by α-Syn PFFs showed a significant increase in the fluorescence intensity ratio of JC-1 polymer to JC-1 monomer, indicating that PCDs could alleviate the damage to mitochondria caused by α-Syn PFFs. The JC-1 detection results indicate that PCDs themselves have no effect on mitochondrial membrane potential, but they can significantly antagonize the decrease in mitochondrial membrane potential induced by α-Syn PFFs, thus exerting a protective effect. Figure 8As shown in Figure c, the effect of PCDs intervention on α-Syn PFFs on intracellular mitochondrial membrane potential was observed under a fluorescence microscope. When the mitochondrial membrane potential was high, the JC-1 polymer produced red fluorescence, and when the mitochondrial membrane potential was low, the JC-1 monomer produced green fluorescence. Figure 8 As shown in d, statistical analysis of the effect of PCDs intervention on α-Syn PFFs on intracellular mitochondrial membrane potential showed that PCDs themselves had no effect on mitochondrial membrane potential, but could significantly antagonize the decrease in mitochondrial membrane potential induced by α-Syn PFFs.

[0094] The above embodiments correspond to the use described in claim 10 of the present invention, wherein the PCD concentration is 0.02 mg / mL falling within the range of 0.01-0.05 mg / mL, the α-Syn PFFs induction concentration is 30 μM falling within the range of 10-50 μM, and the administration method is administration before α-synuclein prefibrillary induction (pretreatment).

[0095] Example 5: PCDs degrade α-Syn pathological aggregates and downregulate phosphorylation levels in SH-SY5Y cells To verify the intervention effect of PCDs on α-Syn pathological aggregation, a cell model was established in the SH-SY5Y cell line using pre-fabricated α-Syn fibers (PFFs). SH-SY5Y cells were seeded in 6-well plates and cultured until the density reached 70-80%. The experiment was divided into four groups: (1) Control group (blank control); (2) PCDs alone treatment group (0.02 mg / mL); (3) α-Syn PFFs treatment group (30 μM); (4) PCDs + α-Syn PFFs combined treatment group (PCDs pretreatment for 24 hours followed by PFFs). All cells were cultured for another 48 hours.

[0096] The presence of α-Syn was analyzed by Western blotting. Cells from each group were collected, and total protein was extracted using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Proteins were separated by SDS-PAGE electrophoresis, and after transfer to a membrane, they were incubated with anti-α-Syn antibody (primary antibody, 1:1000) and anti-p-α-Syn (Ser129) antibody (primary antibody, 1:1000), respectively. HRP-labeled secondary antibody (1:2000) was used for color development, and detection was performed using ECL chemiluminescence.

[0097] The results are as follows Figure 9 a and Figure 9As shown in b: In the Control group and the PCDs-only treatment group, no obvious dimers, trimers, or aggregates were detected. α-Syn mainly existed in monomeric form (~15 kDa), indicating that PCDs themselves do not have aggregation-inducing toxicity. In contrast, the α-Syn PFFs treatment group induced significant pathological aggregation, with a dense diffuse band extending from the dimer to the top of the gel observed, indicating that exogenous PFFs successfully induced endogenous α-Syn to form SDS-resistant oligomers and aggregates. However, in the PCDs and PFFs co-treatment group, this high-molecular-weight diffuse band was significantly weakened, and the signal intensity of dimers and trimers increased significantly, indicating that PCDs can effectively dissociate the PFFs-induced aggregation process.

[0098] In the detection of phosphorylated α-Syn (p-α-Syn, Ser129), the PFFs group showed the strongest phosphorylation signal, while the phosphorylation level in the PCDs+PFFs co-treatment group was significantly reduced, falling to levels close to the control group. This result indicates that PCDs not only physically clear aggregates but also significantly reduce the pathological phosphorylation of α-Syn, suggesting that they may reduce the substrates recognized by kinases or alter protein conformation by blocking aggregate formation, ultimately reducing the overall phosphorylation load. Quantitative analysis further confirmed these observations with statistical significance (***P<0.001).

[0099] Further verification was performed using immunofluorescence staining. Cells in each group were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, incubated overnight with anti-p-α-Syn (Ser129) antibody (primary antibody, 1:400), incubated for 1 hour with Alexa Fluor488-labeled secondary antibody (1:500), stained with DAPI, and observed under a laser confocal microscope.

[0100] The results are as follows Figure 9 c and Figure 9 As shown in Figure d, the cytoplasm of the α-Syn PFFs-treated group was filled with a large number of p-α-Syn positive inclusion bodies (green fluorescence), with high fluorescence intensity and diffuse distribution. In contrast, the cytoplasm of the PCDs+α-Syn PFFs co-treated group was significantly clearer, and the number and size of p-α-Syn inclusion bodies were greatly reduced. Quantitative fluorescence intensity analysis showed a significant decrease (***P<0.001), which directly confirmed the inhibitory effect of PCDs on intracellular protein aggregation.

[0101] In summary, PCDs can effectively inhibit α-Syn PFFs-induced aggregation at the cellular level, accompanied by a significant downregulation of pathological phosphorylation levels.

[0102] The above embodiments correspond to the uses described in claims 8, 10 and 11 of the present invention, wherein the PCD concentration is 0.02 mg / mL falling within the range of 0.01-0.05 mg / mL, the α-Syn PFFs induction concentration is 30 μM falling within the range of 10-50 μM, and the administration method is pretreatment.

[0103] Example 6: Verification of the ability of PCDs to cross the blood-brain barrier in an in vitro model bEnd.3 mouse brain microvascular endothelial cells were cultured in the upper chamber of a Transwell chamber (0.4 μm pore size, polycarbonate membrane) to form a tightly connected monolayer. Monolayer integrity was confirmed by measuring transmembrane resistance (TEER) (TEER > 150 Ω·cm² was considered acceptable). SH-SY5Y cells were seeded in the lower chamber to simulate the brain parenchyma environment.

[0104] The experiment was divided into four groups: (1) Control group (blank control); (2) Cy5.5 labeled control group; (3) Proanthocyanidin monomer (PC) treatment group; and (4) PCDs treatment group. Equal concentrations of PC or PCDs (calculated based on proanthocyanidin equivalents) were added to the upper chamber, and samples were taken from the lower chamber at 15, 30, 60, and 90 minutes. The concentrations of each component in the lower chamber were detected using a fluorescence microplate reader, and the transport efficiency was calculated.

[0105] The results are analyzed as follows: Figure 10 As shown in Figure a, after successful model construction, the TEER values ​​of each group remained stable (>150 Ω·cm) within 90 minutes. 2 This indicates that the bEnd.3 monolayer has good integrity and was not damaged by the experimental treatment.

[0106] like Figure 10 As shown in b, the transport efficiency of PCDs reached approximately 60% at 15 minutes, approximately 75% at 30 minutes, exceeded 90% at 60 minutes, and remained above 90% at 90 minutes. In contrast, the transport efficiency of proanthocyanidin monomers (PC) was less than 20% at all time points and did not increase significantly with time. The transport efficiency of the Cy5.5 control group was close to zero, further validating the barrier integrity of the model.

[0107] The above results show that PCDs exhibit excellent in vitro BBB penetration ability due to their nano-size effect and surface functional group characteristics. Their transport efficiency is 4-5 times that of proanthocyanidin monomers, which solves the key bottleneck of low delivery efficiency of natural polyphenols in the central nervous system.

[0108] The above embodiments correspond to the uses described in claims 8, 10 and 12 of the present invention, wherein the transmembrane transport efficiency of PCDs in the in vitro blood-brain barrier model reaches more than 90% within 60-90 minutes, which is significantly higher than the transport efficiency of proanthocyanidin monomers.

[0109] In summary, this invention successfully synthesized proanthocyanidin carbon dots (PCDs) with characteristic proanthocyanidin functional groups and excellent fluorescence properties. Studies have shown that PCDs not only effectively inhibit pathological fibrosis of α-Syn at the molecular level, promote protofibrillary depolymerization, and significantly reduce the content of their β-sheet structure, but also effectively antagonize α-Syn PFFs-induced neurotoxicity at the cellular level, degrade intracellular pathological aggregates, and downregulate pathological phosphorylation levels. Their protective mechanisms include significantly reducing oxidative stress induced by α-Syn PFFs and alleviating the decline in mitochondrial membrane potential, thereby breaking the vicious cycle of neuronal death. Furthermore, PCDs exhibit excellent transmembrane transport capacity in an in vitro blood-brain barrier model, providing crucial evidence for their in vivo reach of lesions in the central nervous system and their therapeutic effects. These results systematically reveal the potential of PCDs to exert neuroprotective effects through multi-target intervention, providing an effective approach for developing novel therapeutic strategies for neurodegenerative diseases such as Parkinson's disease.

[0110] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification and drawings under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A proanthocyanidin-derived carbon dot, characterized in that, The derived carbon dots are prepared by hydrothermal method using proanthocyanidins as carbon source. The derived carbon dots are spherical nanoparticles with an average diameter of 1-2 nm, a morphological height of 5-15 nm, and a lattice spacing of 0.30-0.38 nm. Their surfaces contain carboxyl and phenolic hydroxyl functional groups and have a π-π conjugated structure.

2. The proanthocyanidin-derived carbon dots according to claim 1, characterized in that, The derived carbon dots are composed of C and O elements, with a C content of 60-80% and an O content of 15-30%. And / or, the surface of the derived carbon dots contains C=O bonds, CH bonds and CO bonds; And / or, the carboxyl and phenolic hydroxyl groups are derived from the functional groups of the proanthocyanidin precursor and are retained.

3. The proanthocyanidin-derived carbon dots according to claim 1 or 2, characterized in that, The derived carbon dots exhibit photoluminescence properties; And / or, the surface of the derived carbon dots is modifiable; And / or, the carbon dots have graphitized carbon cores inside; And / or, the carbon dots are formed by the pyrolysis and carbonization of proanthocyanidins at 180-250°C, and the functional groups of the proanthocyanidin precursor are retained on the surface of the carbon dots after pyrolysis.

4. A method for preparing proanthocyanidin-derived carbon dots according to any one of claims 1-3, characterized in that, include: Step 1: Dissolve proanthocyanidins in a mixed solvent of anhydrous ethanol and ultrapure water, and disperse by ultrasonication; Step 2: Transfer the sonicated solution to a high-pressure reactor and heat at 180-250℃ for 6-16 hours; Step 3: After cooling the reaction solution, filter it, collect the filtrate, remove insoluble residues; dialysis purification, freeze-dry, and store at low temperature.

5. The preparation method according to claim 4, characterized in that, In step 1, the ratio of proanthocyanidins to the mixed solvent is 1:50-200, and the volume ratio of anhydrous ethanol to ultrapure water is 1:2-4. And / or, the ultrasonic dispersion time is 15-60 min; And / or, in step 2, the high-pressure reactor is a high-pressure reactor lined with Teflon; And / or, in step 3, the filtration uses a 0.10-0.45μm microfiltration membrane; And / or, the dialysis purification uses a dialysis membrane with a molecular weight cutoff of 500-2000 Da, the dialysis time is 4-16 h, and the ultrapure water is replaced every 1-4 h; And / or, the low-temperature storage temperature is -80 to 4°C.

6. Use of the proanthocyanidin-derived carbon dots according to any one of claims 1-3 or the proanthocyanidin-derived carbon dots prepared by the method according to any one of claims 4-5 in the preparation of formulations for intervening in the α-synuclein fibrosis process.

7. The use according to claim 6, characterized in that, The preparation is used to treat synucleinosis, which is Parkinson's disease or Lewy body dementia; And / or, the formulation serves as a research tool for studying the mechanisms of protein fibrosis or screening inhibitors; And / or, the proanthocyanidin-derived carbon dots are co-incubated with 0.5-2 mg / mL of α-synuclein pre-fabricated filaments in PBS buffer at 35-40°C and 500-1500 rpm for 1-14 days; the pH of the PBS buffer is 7.0-7.

6.

8. The use according to claim 6 or 7, characterized in that, The proanthocyanidin-derived carbon dots were applied at a concentration of 0.01-0.05 mg / mL to an SH-SY5Y cell model induced by 10-50 μM α-synuclein pre-fibrils, and were administered as a pretreatment. And / or, the formulation is also used for diagnostic imaging based on the photoluminescence properties of the carbon dots; And / or, the formulation is delivered across the blood-brain barrier and targets intracellular α-synuclein filaments; And / or, the transmembrane transport efficiency of the proanthocyanidin-derived carbon dots in an in vitro blood-brain barrier model reaches over 90% within 60-90 minutes; the formulation is also used to downregulate the pathological phosphorylation level of intracellular α-synuclein at the Ser129 site; the proanthocyanidin-derived carbon dots reduce the phosphorylation substrate recognized by kinases or alter protein conformation by degrading intracellular pathological α-synuclein aggregates, thereby reducing the pathological phosphorylation load.

9. A method for evaluating the intervention effect of proanthocyanidin-derived carbon dots on the pathological aggregation of α-synuclein in a cell model, characterized in that, Includes the following steps: Step 1: Establish a pathological aggregation model by treating SH-SY5Y cells with α-synuclein pre-fabricated filaments; Step 2: Co-treat the cells with proanthocyanidin-derived carbon dots and α-synuclein pre-fabricated fibers; Step 3: Detect the expression levels of intracellular α-synuclein monomers, oligomers, aggregates, and phosphorylated α-synuclein by Western blotting; Step 4: Detect the distribution and fluorescence intensity of phosphorylated α-synuclein-positive inclusion bodies in cells by immunofluorescence staining; Step 5: Quantitatively analyze the inhibitory effect of proanthocyanidin-derived carbon dots on the pathological aggregation and phosphorylation of α-synuclein.

10. A method for evaluating the ability of proanthocyanidin-derived carbon dots to cross the blood-brain barrier, characterized in that, Includes the following steps: Step 1: Culture bEnd.3 cells in the upper chamber of a Transwell chamber to form a tight junction monolayer; Step 2: Seed SH-SY5Y cells in the lower chamber; Step 3: Add proanthocyanidin-derived carbon dots or proanthocyanidin monomers to the upper chamber and confirm the integrity of the monolayer by detecting the transmembrane resistance; Step 4: Determine the transport efficiency of proanthocyanidin-derived carbon dots or proanthocyanidin monomers in the lower chamber at different time points. Step 5: Compare the differences in transmembrane transport between proanthocyanidin-derived carbon dots and proanthocyanidin monomers.

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