Near-infrared carbon dots and application thereof in preparation of Alzheimer disease diagnosis and treatment preparation

Abstract

The invention discloses a near-infrared carbon dot and application of the near-infrared carbon dot in preparation of an Alzheimer disease diagnosis and treatment preparation. The near-infrared carbon dot is obtained by performing solvothermal reaction on glutathione and formamide at 140-150 DEG C for 3-5 hours and then performing dialysis. According to the probe, through accurate structural design, a synergistic effect is formed by morphology, a crystal structure, optical performance, strong negative surface charge and surface functional group characteristics, a structural basis for implementing multi-target intervention aiming at an AD core pathogenic mechanism is jointly formed, and a performance support is provided for a near-infrared imaging function. Wherein strong negative surface charges directly inhibit Abeta42 aggregation through electrostatic repulsion, a two-dimensional layered graphite-like structure promotes rapid transfer of surface electrons, carboxyl, sulfydryl and other functional groups synergistically improve Cu < 2 + > chelation and ROS removal efficiency, and the carboxyl, sulfydryl and other functional groups form a synergistic network of physical repulsion-chemical chelation-electron transfer. A key foundation is laid for developing a next-generation AD targeted nano treatment / diagnosis material.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, specifically to a near-infrared carbon dot and its application in the preparation of Alzheimer's disease diagnostic and therapeutic agents, which can be used to synergistically block the pathological cascade of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is one of the most challenging neurodegenerative diseases, and its core pathological process is driven by the abnormal aggregation of amyloid-β (Aβ) and metal ions (especially Cu). 2+ Driven by the synergistic effect of abnormal enrichment. Specifically, Cu 2+ It exacerbates nerve damage through a dual pathogenic pathway: on the one hand, it binds to the His6, His13, and His14 sites of the Aβ peptide to form Aβ-Cu. 2+ The complex directly induces Aβ aggregation and fibrosis; on the other hand, it catalyzes the massive generation of neurotoxic reactive oxygen species (ROS), exacerbating oxidative stress, while interfering with the function of antioxidant enzymes such as superoxide dismutase (SOD), damaging mitochondrial integrity, and ultimately inducing neuronal apoptosis. Although drugs targeting Aβ, such as Aducanumab, have made breakthroughs in mechanism, their limited clinical efficacy and high cost highlight the urgent need to develop multifunctional intervention tools that simultaneously intervene in Aβ aggregation, metal ion toxicity, oxidative stress, and mitochondrial damage, and also have pathological imaging capabilities.

[0003] Currently, building such a multifunctional platform integrating diagnosis and treatment still faces significant challenges. Existing strategies mostly target single pathological stages: traditional Aβ inhibitors are ineffective at dissociating already formed fibers; common metal chelators lack selectivity and may disrupt physiological metal homeostasis; exogenous antioxidants often fail to target damaged organelles (such as mitochondria) and have short-lived effects. In addition, most near-infrared imaging probes have limited functionality, making it difficult to simultaneously achieve treatment and real-time monitoring.

[0004] In recent years, although carbon dots (CDs) have shown advantages such as good biocompatibility and ease of functionalization, their application in AD has mostly been limited to the exploration of single functions, and has not yet been able to effectively integrate "multi-target synergistic intervention" and "pathological visualization monitoring". Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing probes and develop a new probe for AD "Aβ". 42 Aggregate-Cu 2+ NGN-CDs, a multifunctional nanoprobe targeting the "toxicity-oxidative stress-mitochondrial damage" cascade pathogenesis mechanism, possess unique characteristics including morphology, crystal structure, optical properties, strong negative surface charge, and surface functional groups. These characteristics collectively constitute its ability to target the core pathogenesis mechanism of Alzheimer's disease (Aβ aggregation, Cu...). 2+This provides the structural basis for multi-target intervention (due to abnormalities and oxidative stress) and offers performance support for near-infrared imaging. Specifically, strong negative surface charges directly inhibit Aβ through electrostatic repulsion. 42 Aggregation is one of the key physicochemical properties for realizing the functionality of NGN-CDs materials. These characterization results fully validate the rationality of the material design and lay the foundation for its subsequent applications in anti-amyloid formation and Cu... 2+ Research on its bioactivities, including chelation, anti-oxidative stress, and cell protection, has laid a solid foundation. It provides an integrated platform for synergistic intervention and visual monitoring to address amyloid-related neuropathological problems, and also lays a crucial foundation for developing next-generation AD-targeted nanomaterials for therapy and diagnosis.

[0006] To achieve the above objectives, the technical solution specifically adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a near-infrared carbon dot, wherein the surface of the carbon dot contains carboxyl and thiol functional groups, has a two-dimensional layered graphite-like structure, a negative Zeta potential, and exhibits strong near-infrared emission at 685 nm; the near-infrared carbon dot is prepared by the following method:

[0008] Glutathione was dissolved in formamide and subjected to a solvothermal reaction at 140-150℃ for 3-5 hours. Then, it was transferred into a dialysis bag for dialysis purification to obtain the near-infrared carbon dots, denoted as NGN-CDs.

[0009] Further purification was performed using dialysis bags at a concentration of 1000-3500 Da.

[0010] Furthermore, the Zeta potential is -20 mV to -25 mV, preferably -22.8 mV.

[0011] The principle of this invention is to reduce Aβ by using a strong negative surface charge. 42 The probability of monomer / aggregate collision and binding is enhanced by the synergistic effect of two-dimensional layered structure and functional groups such as carboxyl and thiol groups on Cu. 2+ Chelating ability and antioxidant activity, along with near-infrared luminescence properties, offer potential for pathological visualization. Systematic characterization and biological experiments confirm that NGN-CDs have a chelating effect on Cu. 2+ It exhibits high binding affinity, significantly inhibits its peroxidase-like activity, and effectively blocks Aβ. 42 It inhibits fibrillation and breaks down pre-formed fibrils, reducing fibroblast toxicity; in the SH-SY5Y cell model, it can significantly alleviate H2O2 / Aβ. 42 -Cu 2+ Induced oxidative stress and mitochondrial damage; simultaneously exhibits strong near-infrared fluorescence emission at 685 nm, possessing Aβ plaques and Cu... 2+ Imaging capability for enriched regions.

[0012] Secondly, the present invention provides the application of the above-mentioned near-infrared carbon dots in the preparation of Alzheimer's disease diagnostic and therapeutic agents.

[0013] Furthermore, the therapeutic agent is used to synergistically block the pathological cascade of Alzheimer's disease, and the synergistic blocking includes at least one of the following mechanisms:

[0014] (1) Suppressing Aβ through the electrostatic repulsion of negative charges on the carbon dot surface 42 Protein aggregation and fibrosis;

[0015] (2) Cu chelation through functional groups on the surface of carbon dots 2+ Inhibit Cu 2+ Induced Aβ 42 Aggregation and Cu 2+ Catalytic generation of reactive oxygen species;

[0016] (3) It removes reactive oxygen species (ROS) in cells and protects mitochondrial membrane potential through the electron transfer ability of carbon dots.

[0017] Furthermore, the therapeutic agent has an effect on Aβ plaques or Cu in the brain. 2+ The ability to perform near-infrared fluorescence imaging in enriched regions.

[0018] Thirdly, the present invention provides a pharmaceutical composition for intervening in the pathological process of Alzheimer's disease, wherein the active ingredient comprises at least the aforementioned near-infrared carbon dots.

[0019] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention constructs near-infrared carbon dots (NGN-CDs) with unique structures by controlling the reaction temperature and time. The temperature range provided by this invention can precisely control the degree of graphitization and the exposure of surface functional groups of the carbon dots, avoiding functional group decomposition or excessive graphitization caused by high temperature, while the short reaction time can retain more active functional groups (carboxyl groups, thiol groups) and ensure the crystal integrity of the carbon dots.

[0022] The near-infrared carbon dots (NGN-CDs) obtained through the above-mentioned condition regulation exhibit rapid surface electron transfer in their two-dimensional layered structure. Surface carboxyl and thiol groups directly participate in the Redox reaction, enabling dose-dependent scavenging of ∙OH, DPPH∙, and ABTS. ∙+ Furthermore, due to its mitochondrial targeting properties (PCC=0.96), it can concentrate its effects at the damage site, directly protecting the mitochondrial membrane potential; simultaneously, it forms a stable coordination complex through N / Cu and carboxylate / Cu interactions, binding constant K. b=7.73×10 3 It can not only prevent Cu 2+ Driven Aβ 42 Aggregation can also inhibit Cu 2+ The peroxidase-like activity eliminates the source of ROS catalysis, forming a dual effect of "chelation-enzyme activity inhibition".

[0023] In NGN-CDs, the strong negative charge (-22.8 mV) reduces Aβ through electrostatic repulsion. 42 The collisional assembly of monomers / oligomers, while the two-dimensional layered structure can be inserted into Aβ 42 By disrupting the stability of the fiber's β-fold structure, a dual function of "prevention + reversal" can be achieved, unlike traditional materials that can only inhibit aggregation.

[0024] Furthermore, NGN-CDs achieve strong near-infrared emission at 685 nm based on their intrinsic carbon skeleton structure, with minimal fluorescence fluctuation (<4% within 9 min). They achieve structural unity of therapeutic and imaging functions without additional modification, avoiding the drawbacks of traditional multi-component composite probes such as easy dissociation and poor stability. This facilitates real-time visualization of AD-related intracellular sites (such as damaged mitochondria and Aβ). 42 Aggregates). Attached Figure Description

[0025] Figure 1 This is a flowchart illustrating the principle of the present invention;

[0026] Figure 2 The image shows a transmission electron microscope (TEM) image of the probe invented in Example 1.

[0027] Figure 3 This is a 3D fluorescence scan of the probe invented in Example 1;

[0028] Figure 4 For different Cu in Example 2 2+ Fluorescence spectrum of the probe at the specified concentration;

[0029] Figure 5 In Example 2, NGN-CDs inhibit Cu 2+ Induced Aβ 42 Protein aggregation and depolymerization of Aβ 42 TEM image of aggregate;

[0030] Figure 6 These are confocal images at different time points after NGN-CDs and SH-SY5Y cells were co-incubated for 2 h in Example 2.

[0031] Figure 7 For example, NGN-CDs and H2O2 / (Aβ) in Example 2 42 +Cu2+ )1 Laser confocal microscope image of DCFH-DA / JC-1 stained SH-SY5Y cells after co-incubation. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0033] Example 1

[0034] The principle of this invention is as follows Figure 1 As shown.

[0035] 1.05 g of L-GSH was dissolved in 30 mL of formamide by sonication and then transferred to a polytetrafluoroethylene high-pressure reactor. The reactor was placed in a constant temperature oven at 140 °C for 4 h. After the reaction, the filtered solution was diluted and transferred to a 3500 Da dialysis bag for dialysis purification for one week. The resulting NGN-CDs were obtained by lyophilization. The TEM image and fluorescence 3D scan image of the carbon dots are shown below. Figure 2 , 3 As shown, the size is around 2-4 nm, and it produces strong near-infrared fluorescence emission at 685 nm when the excitation wavelength is 420 nm.

[0036] Example 2

[0037] (a) Probe and Cu 2+ Combined ability test

[0038] The probe of this invention was subjected to different concentrations of Cu. 2+ After co-incubation at room temperature for 15 min, the fluorescence intensity (λ) was measured. ex =420 nm, λ em =685 nm). For example... Figure 4 As shown, with Cu 2+ As the concentration increases, the fluorescence intensity of the probe gradually decreases. A linear curve was plotted based on the Benesi-Hildebrand equation, and the binding constant K was calculated. b =7.73×10 3 This demonstrates its high affinity chelating ability.

[0039] (ii) Probe suppression of Cu 2+ Induced Aβ 42 Protein aggregation and deaggregation test

[0040] Two sets of experiments were set up: ① Aβ 42 With Cu 2+ ;② Aβ 42 With Cu 2+Both the probe NGN-CDs of this invention and the incubator were incubated in PBS at 37 °C for 4 days. TEM images were taken after incubation. Figure 5 The results showed that group ② had no significant Aβ. 42 Fiber formation; additional prefabricated Aβ 42 The co-incubation of fibers with NGN-CDs showed that the fiber structure was destroyed, demonstrating its bidirectional "inhibition-depolymerization" function.

[0041] (III) Probe Imaging Stability Test

[0042] After co-incubating NGN-CDs with SH-SY5Y cells for 2 h, continuous imaging was performed for 9 min. Figure 6 As shown, the fluorescence intensity did not change significantly (fluctuation < 4%) during the 9-minute observation period, indicating that its near-infrared fluorescence has excellent temporal stability and meets the requirements for real-time imaging.

[0043] (iv) Verification of probe inhibition of intracellular oxidative stress and mitochondrial protection

[0044] Human neuroblastoma cells SH-SY5Y were placed in MEM medium containing 5% fetal bovine serum (FBS) and cultured at 37 ℃ and 5% CO2. Cells were seeded at a density of 20,000 cells per well in confocal microplates. After 24 h of culture, H2O2 / (Aβ) was added to each well. 42 +Cu 2+ 1. Cells were co-cultured with NGN-CDs for 24 h. Cells were then washed with MEM, and ROS levels were detected by DCFH-DA staining, while mitochondrial membrane potential was detected by JC-1 staining. Figure 7 As shown, compared with the blank control group (CT), the injury model group (H2O2 group and Aβ group) 42 +Cu 2+ The cells in the first group exhibited strong green fluorescence, indicating extremely high intracellular ROS levels. However, after treatment with NGN-CDs, the green fluorescence in the cells was significantly reduced, indicating that the ROS level in the NGN-CDs-treated group was significantly reduced and the mitochondrial membrane potential remained stable, demonstrating the synergistic effect of its antioxidant and mitochondrial protective effects.

[0045] Based on the above experimental results, it can be seen that the carbon dots prepared by this invention have a size of <10 nm and have abundant hydrophilic functional groups on their surface. Therefore, it is expected that they can penetrate the blood-brain barrier and have application prospects in Alzheimer's disease diagnostic and therapeutic agents.

[0046] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A near-infrared carbon dot, characterized in that, The carbon dots have carboxyl and thiol functional groups on their surface, possess a two-dimensional layered graphite-like structure, a negative Zeta potential, and near-infrared emission at 685 nm. These near-infrared carbon dots are prepared using the following method: Glutathione was dissolved in formamide and subjected to a solvothermal reaction at 140-150℃ for 3-5 hours. Then, it was transferred into a dialysis bag for dialysis purification to obtain the near-infrared carbon dots, denoted as NGN-CDs.

2. The near-infrared carbon dot according to claim 1, characterized in that, Purification was performed using dialysis bags with a capacity of 1000-3500 Da.

3. The near-infrared carbon dot according to claim 1, characterized in that, The zeta potential is -20 mV to -25 mV.

4. The use of near-infrared carbon dots as described in any one of claims 1-3 in the preparation of Alzheimer's disease diagnostic and therapeutic agents.

5. The application according to claim 4, characterized in that, The therapeutic agent is used to synergistically block the pathological cascade of Alzheimer's disease, and the synergistic blocking includes at least one of the following mechanisms: (1) Suppressing Aβ through the electrostatic repulsion of negative charges on the carbon dot surface 42 Protein aggregation and fibrosis; (2) Cu chelation through functional groups on the surface of carbon dots 2+ Inhibit Cu 2+ Induced Aβ 42 Aggregation and Cu 2+ Catalytic generation of reactive oxygen species; (3) It removes reactive oxygen species in cells and protects mitochondrial membrane potential through the electron transfer ability of carbon dots.

6. The application according to claim 4, characterized in that, The therapeutic agent has effects on Aβ plaques or Cu in the brain. 2+ The ability to perform near-infrared fluorescence imaging in enriched regions.

7. A pharmaceutical composition for intervening in the pathological process of Alzheimer's disease, characterized in that, Its active ingredient contains at least the near-infrared carbon dots as described in any one of claims 1-3.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

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