CN114377033B - Preparation method and application of inhibitor carbon dot for inhibiting amyloid beta protein aggregation - Google Patents

Preparation method and application of inhibitor carbon dot for inhibiting amyloid beta protein aggregation Download PDF

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CN114377033B
CN114377033B CN202210180468.8A CN202210180468A CN114377033B CN 114377033 B CN114377033 B CN 114377033B CN 202210180468 A CN202210180468 A CN 202210180468A CN 114377033 B CN114377033 B CN 114377033B
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董晓燕
林小丁
余林玲
孙彦
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Abstract

The invention relates to a preparation method and application of an inhibitor carbon dot for inhibiting amyloid beta protein aggregation. Respectively dissolving a micromolecular amyloid beta protein aggregation inhibitor and a doping agent containing heteroatoms in deionized water, heating in a water bath kettle to dissolve completely, transferring a solution with the volume not exceeding 2/3 of that of the reaction kettle into a polytetrafluoroethylene reaction kettle, putting into an electrothermal blowing drying box, and completing the reaction by a hydrothermal method; and (3) putting the reaction solution into a dialysis bag with a molecular weight cut-off for dialysis, removing redundant reactants and impurities, and concentrating the obtained solution by a rotary steaming method and a freeze drying method to obtain inhibitor carbon dot powder. Inhibitor carbon dots can effectively inhibit Abeta at low concentration 40 Is significantly reduced in Abeta 40 Generation of beta-sheet structures during aggregation. Inhibitor carbon dots are capable of inhibiting aβ expressed in CL2006 in vivo 42 Toxicity generated by aggregation induction, prolonged CL2006 life, is an ideal inhibitor of amyloid beta aggregation.

Description

Preparation method and application of inhibitor carbon dot for inhibiting amyloid beta protein aggregation
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a preparation method and application of an inhibitor carbon dot for inhibiting amyloid beta protein aggregation.
Background
Alzheimer's Disease (AD) is a slowly progressive degenerative Disease of the nervous system, the most common form of dementia, and patients with clinical symptoms such as memory decline, cognitive dysfunction, and disorientation (Lancet, 2006, 368:387-403). AD is the most common cause of senile dementia, and about 5000 or more tens of thousands of people are affected by it worldwide in 2020, 8300 tens of thousands by 2030 and 1.52 million by 2050 (Molecules, 2021, 26 (6)). At present, the pathogenesis of AD is not yet clear, and some scholars have proposed several hypotheses for the pathogenesis of AD, including the inflammatory hypothesis, the tau hypothesis, the Amyloid-beta protein (Abeta) hypothesis and the cholinergic hypothesis, wherein the Amyloid-beta cascade hypothesis (Abeta cascade hypothesis, ACH) is widely accepted (Science of The Total Environment,2020, 700:134836). ACH considers that accumulation of amyloid beta peptide in brain occurs inEarly stages of AD, which can then lead to the formation of Senile Plaques (SPs) and subsequent neurofibrillary tangles (NFTs), cause neuronal cell death, and ultimately dementia (The Protein Journal,2019,38 (4): 425-434). Amyloid beta protein is a small peptide composed of 39-43 amino acids, which is produced by cleavage of amyloid precursor protein (Amyloid Precursuor Protein, APP) by beta-secretase and gamma-secretase, the most common form of amyloid beta protein being 40 amino acids aβ 40 And a 42 amino acid aβ 42 Two (The Protein Journal,2019,38 (4): 425-434). Amyloid beta proteins aggregate within the brain of patients, self-assemble into soluble amyloid beta oligomers and insoluble amyloid beta fibers, and exert toxic and oxidative stress on neuronal cells, even gradually leading to neuronal synaptic dysfunction, leading to neuronal death and ultimately to injury to the patient's nervous system (Science, 2002, 297 (5580): 353-356). Thus, designing a suitable amyloid β protein inhibitor, utilizing the interaction between the inhibitor and amyloid β protein, blocking the amyloid β protein fiber formation pathway or disrupting the β -sheet in the amyloid β protein fiber structure to form low-toxic amorphous aggregates, may alleviate the symptoms of AD.
The current amyloid beta protein inhibitors are small molecule inhibitors, polypeptide inhibitors, protein inhibitors and nanoparticle inhibitors. The small molecule inhibitors have simple structure, obvious effect and good BBB penetrating performance, but most small molecule inhibitors have poor water solubility and low specificity, so that the bioavailability is low (ACS Chemical Neuroscience,2019, 10 (8): 3366-3374); polypeptide inhibitors have the advantages of small molecular weight, ease of synthesis, no immunogenicity and strong specificity, but are easily self-aggregating, have a high effective concentration and do not readily pass the BBB (ACS Chemical Neuroscience,2017,9 (2): 198-210); protein inhibitors, while having good biocompatibility, are difficult to pass the BBB (ACS Chemical Neuroscience,2019, 10 (8): 3366-3374). In contrast, nanoparticle inhibitors have a large specific surface area and simple synthesis although the specificity to amyloid beta protein is not high, and other inhibitors (such as small molecules, peptides, proteins and the like) can be introduced through surface modification, so that the nanoparticle inhibitors are inhibitors with wide research and development prospects. Nanomaterial variety is diverse, and Quantum Dots (QDs) having unique physicochemical properties have attracted extensive attention in the scientific community in recent years due to their medical applications. Among them, carbon quantum dots (CDs) have unique structures and properties in which conjugated pi electrons can undergo pi-pi stacking interactions with aromatic rings of amino acid residues in amyloid polypeptides, thereby affecting the amyloid beta protein aggregation pathway (Adv mate, 2013, 25 (28): 3780-801). Several studies have reported that CDs have a significant inhibitory effect on amyloidosis (nanoscales, 2019, 11 (46): 22387-22397) and can be used as detection probes for amyloid (Protein and Peptide Letters, 2019). In addition, CDs have the characteristics of simple preparation method, easy functionalization, low toxicity, high biocompatibility, adjustable optical characteristics and the like. Various small molecules, polymers or biomass have been screened as precursors for the synthesis of CDs, but most CDs currently have little or no inhibitory effect on amyloid β protein aggregation.
Many small organic molecule inhibitors have good inhibition effect on amyloid beta protein aggregation, such as Epigallocatechin-3-gallate (EGCG), curcumin, resveratrol, brazilin and the like, but most small molecule inhibitors have poor water solubility and low specificity.
Disclosure of Invention
The invention designs based on the organic small molecule amyloid beta protein inhibitor, creatively proposes to utilize the organic small molecule amyloid beta protein inhibitor as a raw material to synthesize carbon dots, so as to enhance the inhibition effect of the carbon dots, improve the defects of the small molecule inhibitor and generate the multifunctional carbon dot inhibitor. Polyphenols are widely present in foods, and many studies have shown that natural polyphenols inhibit amyloid fibril formation, exhibiting good anti-amyloidosis (Molecular nutrition & food research,2015, 59:8-20). EGCG is a green natural plant polyphenol capable of significantly inhibiting the formation of amyloid beta protein aggregates. In addition, EGCG has catechol structure, and research shows that the polyphenol compound has wide biological activity including metal chelating, anti-inflammatory, anticancer, antimicrobial and antioxidant activity, etc. (The Science of the Total Environment,2020,700: 134836.1-134836.11), and the phenol compound with catechol structure can inhibit aggregation of amyloid beta protein and human islet amyloid polypeptide (hIAPP), and has antioxidant activity and protective effect (Natural Product Communications,2019,14 (5): 1934578X 1984303). Studies have shown that the incorporation of heteroatoms has a great influence on the properties of the carbon dots, o-phenylenediamine (oPD) is a commonly used nitrogen dopant (Small, 2019,15 (48): 1901507), and o-phenylenediamine containing nitrogen heteroatoms can improve the surface emission state of the carbon dots, thereby changing the fluorescent properties of the carbon dots; and the o-phenylenediamine is used as a nitrogen source to ensure that amino groups are kept on the surface of a carbon point, so that the surface charge of the carbon point can be improved, electrostatic interaction between positively charged amino groups on the surface of the carbon point and negatively charged amyloid beta protein can be generated, and the effect of inhibiting amyloid beta protein aggregation is achieved (Small, 2020,16).
The invention creatively designs a small molecular amyloid beta protein inhibitor, and provides a method for synthesizing carbon dots by using an organic small molecular amyloid beta protein inhibitor as a raw material so as to enhance the inhibition effect of the carbon dots, improve the defects of the small molecular inhibitor and generate a multifunctional carbon dot inhibitor. The carbon-containing amyloid beta aggregation small molecule inhibitor is used as a precursor, and partial functional groups of the carbon-containing amyloid beta aggregation small molecule inhibitor are probably an effective method for improving the interaction between carbon dots and amyloid beta proteins and enhancing the inhibition of amyloid beta protein aggregation by the carbon dots, and is also an effective strategy for regulating CDs to play a multifunctional role. Therefore, the invention provides a new research view for the research and development of multifunctional high-efficiency amyloid beta protein aggregation inhibitors.
Therefore, the invention is innovatively designed based on the micromolecular amyloid beta protein inhibitor, utilizes the micromolecular amyloid beta protein aggregation inhibitor as a main carbon source, introduces a doping agent containing hetero atoms to obtain inhibitor carbon points (the carbon points are named as E-PCDs), has higher affinity with the amyloid beta protein, and can effectively inhibit the aggregation of the amyloid beta protein under low concentration. This suggests that using a carbonaceous amyloid β protein aggregation small molecule inhibitor as a precursor, using a portion of its functional groups, may be an effective method for enhancing the interaction of carbon dots with amyloid β proteins, enhancing the inhibition of amyloid β protein aggregation by carbon dots, and also an effective strategy for modulating the development of multiple functions by CDs. Therefore, the invention provides a new research view for the research and development of multifunctional high-efficiency amyloid beta protein aggregation inhibitors.
The invention aims to provide an inhibitor carbon point with a strong amyloid beta protein aggregation inhibiting function, a preparation method and application thereof in inhibiting amyloid beta protein aggregation, and structural expression is shown in figure 1. The inhibitor carbon dot has remarkable inhibition effect on amyloid beta protein aggregation and can relieve Abeta 40 Induced cytotoxicity, eliminates amyloid plaques in AD model caenorhabditis elegans CL2006 and prolongs nematode life, and the specific technical scheme is as follows:
the carbon point synthesized by using the small molecular amyloid beta protein inhibitor takes the small molecular amyloid beta protein aggregation inhibitor as a main carbon source and is doped with a doping agent containing hetero atoms, and the inhibitor carbon point (E-PCDs) is obtained by a green one-step hydrothermal method, and the reaction process is shown in figure 2.
The technical scheme of the invention is as follows:
the invention provides an inhibitor carbon point for inhibiting amyloid beta protein aggregation, which is obtained by taking a small molecular amyloid beta protein aggregation inhibitor as a main carbon source and doping a doping agent containing hetero atoms by utilizing a carbon point synthesized by an organic small molecular amyloid beta protein inhibitor and adopting a one-step hydrothermal method.
The preparation method of the inhibitor carbon dot for inhibiting amyloid beta protein aggregation comprises the following steps:
1) The small molecule amyloid beta protein aggregation inhibitor and the doping agent containing hetero atoms are respectively mixed according to the following ratio of 2-120: dissolving 10-100 molar concentration ratio in deionized water, heating in a water bath kettle to dissolve completely, transferring the solution with volume not exceeding 2/3 of that of the reaction kettle into a polytetrafluoroethylene reaction kettle, placing into an electrothermal blowing drying box, and reacting for 4-12h at 80-200 ℃ by a hydrothermal method to complete the reaction;
2) The reaction solution in the step 1) is put into a dialysis bag with the molecular weight cut-off of 100-5000Da, dialyzed for 12-72 hours in deionized water, then dialyzed for 12-72 hours in 50-100% ethanol, the dialysate is replaced every 4-6 hours, redundant reactants and impurities are removed, and the obtained solution is concentrated through a rotary steaming method and a freeze drying method, so that inhibitor carbon dot powder is obtained.
The micromolecular amyloid beta protein aggregation inhibitor comprises EGCG, curcumin, brazilin, resveratrol, human serum albumin or other micromolecular inhibitors.
The heteroatom-containing dopant includes o-phenylenediamine, cysteine, or other heteroatom-containing dopant.
The molar concentration ratio of the small molecular amyloid beta protein aggregation inhibitor to the heteroatom-containing dopant is preferably 10-75:40-50.
Preferably, the reaction condition of the hydrothermal method is 120-200 ℃ and the reaction time is 10-12h.
Preferably, the dialysis bag has a molecular weight cut-off of 500-3000Da and a dialysis time of 24-48 hours.
The solution obtained is preferably dialyzed against a 0.22 μm nylon membrane before spin-steaming at a temperature of 50-60 ℃.
The invention relates to an application of an inhibitor carbon point for inhibiting amyloid beta protein aggregation in preparing a medicament for treating Alzheimer's disease.
The preferable volume of the reaction kettle is 25mL and 50mL, and the corresponding reaction solvent system is 10mL and 20mL of deionized water respectively.
The inhibitor carbon point with the function of inhibiting amyloid beta protein aggregation, the preparation method and the application thereof in inhibiting amyloid beta protein aggregation have the following advantages:
first, inhibitor carbon sites are effective at inhibiting aβ at low concentrations 40 Is significantly reduced in Abeta 40 Generation of beta-sheet structures during aggregation.
Second, inhibitor carbon sites can alter aβ 40 Inhibit the formation of amyloid fibrils.
Thirdly, the inhibitor carbon dots have good biocompatibility and can be relievedAβ 40 Induced cytotoxicity.
Fourth, the inhibitor carbon dots are capable of inhibiting amyloid plaque formation in the AD model caenorhabditis elegans CL2006, clearing CL2006 dyskinesia.
Fifth, the inhibitor carbon dot is capable of inhibiting aβ expressed in CL2006 in vivo 42 Toxicity generated by aggregation induction, prolonged CL2006 life, is an ideal inhibitor of amyloid beta aggregation.
The invention provides an inhibitor carbon point which can be used for inhibiting amyloid beta protein aggregation, in the invention, a small molecular amyloid beta protein aggregation inhibitor and a nitrogen heteroatom-containing dopant are taken as examples, the small molecular amyloid beta protein aggregation inhibitor is taken as a main carbon source, the nitrogen heteroatom-containing dopant is added, and the inhibitor carbon point (E-PCDs) is synthesized by a green one-step hydrothermal method. E-PCDs have the ability to inhibit aggregation of amyloid beta proteins, with varying concentrations of E-PCDs and Abeta 40 Aβ can be reduced in concentration-dependent relationship in co-culture 40 The E-PCDs can inhibit about 80% of Abeta at a low concentration of 2.5 mug/mL 40 Fibers, demonstrating the pair Aβ of E-PCDs 40 The beta-sheet structure is aggregated to have a strong inhibition effect; the circular dichroism spectrum proves that E-PCDs can effectively inhibit Abeta in a concentration-dependent relation 40 The generation of parallel beta-sheet conformation reduces beta-sheet content, and can lead Abeta at high concentration of 80 mug/mL 40 Maintaining an irregular coiled structure; AFM experiments show that E-PCDs can significantly reduce Abeta 40 Fiber production and change of Abeta 40 Is a aggregated morphology of (a); cell experiments prove that the E-PCDs have good biocompatibility and can relieve Abeta 40 Induced cytotoxicity, saving cell viability to more than 80%; in vivo nematode experiments prove that the E-PCDs can inhibit the formation of amyloid plaques in CL2006, and prolong the service life of CL2006 by 6 days.
The carbon dot E-PCDs are applied to preparation of medicines for treating Alzheimer's disease.
Drawings
Fig. 1: schematic structural diagram of E-PCDs.
Fig. 2: schematic of the synthetic pathways of E-PCDs.
Fig. 3: example 8E-PCDs and Abeta at various concentrations 40 ThT fluorescence kinetics in co-culture.
Fig. 4: different concentrations of E-PCDs and Abeta in example 9 40 Circular Dichroism (CD) spectrogram of the culture after 72h co-culture
Fig. 5: E-PCDs and Abeta at various concentrations in example 10 40 Topography of the culture after 120h co-culture.
Fig. 6: different concentrations of E-PCDs and Abeta in example 11 40 Cell activity profile after co-culture with SH-SY5Y cells.
Fig. 7: fluorescence microscopy of E-PCDs to clear amyloid plaques in CL2006 in example 12.
Fig. 8: survival of CL2006 after co-cultivation of E-PCDs with CL2006 in example 13.
Detailed Description
The invention will be further illustrated with reference to specific examples.
Example 1: preparation process of inhibitor EGCG carbon dots
1) The small molecule amyloid beta protein aggregation inhibitor EGCG and the nitrogen heteroatom dopant o-phenylenediamine (oPD) are mixed according to the following ratio of 75:50 molar concentration ratio is dissolved in deionized water, heated in a water bath kettle to be completely dissolved, then 10mL of solution is transferred into a 25mL polytetrafluoroethylene reaction kettle, and the solution is placed into an electrothermal blowing drying box to react for 10 hours at 200 ℃ by a hydrothermal method to complete the reaction;
2) The reaction solution in the step 1) is put into a dialysis bag with the molecular weight cut-off of 500Da, dialyzed for 48 hours in deionized water, then dialyzed for 48 hours in 50% ethanol, the dialysate is replaced every 6 hours, redundant reactants and impurities are removed, and the obtained solution is concentrated by a rotary evaporation method and a freeze drying method at 55 ℃ to obtain inhibitor EGCG carbon dots (named 75E-PCDs) powder.
Example 2:
1) The small molecule amyloid beta protein aggregation inhibitor EGCG and the nitrogen heteroatom dopant o-phenylenediamine (oPD) are mixed according to the following weight ratio of 10:50 molar concentration ratio is dissolved in deionized water, heated in a water bath kettle to be completely dissolved, then 10mL of solution is transferred into a 25mL polytetrafluoroethylene reaction kettle, and the solution is placed into an electrothermal blowing drying box to react for 10 hours at 120 ℃ by a hydrothermal method to complete the reaction;
2) The reaction solution in the step 1) is put into a dialysis bag with the molecular weight cut-off of 100Da, dialyzed for 24 hours in deionized water, then dialyzed for 24 hours in 50% ethanol, the dialysate is replaced every 6 hours, redundant reactants and impurities are removed, and the obtained solution is concentrated by a rotary evaporation method and a freeze drying method at 55 ℃ to obtain inhibitor EGCG carbon dots (named as 10E-PCDs) powder.
Example 3:
1) The small molecule amyloid beta protein aggregation inhibitor EGCG and the nitrogen heteroatom dopant o-phenylenediamine (oPD) are mixed according to the following steps of 120: dissolving 50 molar concentration ratio in deionized water, heating in a water bath kettle to dissolve completely, transferring 20mL of solution into a 50mL polytetrafluoroethylene reaction kettle, placing into an electrothermal blowing drying box, and reacting for 12 hours at 180 ℃ by a hydrothermal method to complete the reaction;
2) The reaction solution in the step 1) is put into a dialysis bag with the molecular weight cut-off of 1000Da, dialyzed for 48 hours in deionized water, then dialyzed for 48 hours in 50% ethanol, the dialysate is replaced every 6 hours, redundant reactants and impurities are removed, and the obtained solution is concentrated by a rotary evaporation method and a freeze drying method at 55 ℃ to obtain inhibitor EGCG carbon dots (named as 120E-PCDs) powder.
Example 4: preparation process of inhibitor curcumin carbon dots
1) Curcumin, a small molecule amyloid beta protein aggregation inhibitor, and cysteine (Cys), a sulfur-containing heteroatom dopant, were mixed according to a ratio of 40:45 molar concentration ratio is dissolved in deionized water, heated in a water bath kettle to be completely dissolved, then 20mL of solution is transferred into a 50mL polytetrafluoroethylene reaction kettle, and the solution is placed into an electrothermal blowing drying box to react for 11 hours at 150 ℃ by a hydrothermal method to complete the reaction;
2) And (2) putting the reaction solution in the step (1) into a dialysis bag with the molecular weight cut-off of 1500Da, dialyzing in deionized water for 36 hours, then putting into absolute ethyl alcohol for dialyzing for 36 hours, changing the dialysate every 5 hours, removing redundant reactants and impurities, and concentrating the obtained solution by a rotary evaporation method and a freeze drying method at 60 ℃ to obtain the inhibitor curcumin carbon dot powder.
Example 5: preparation process of resveratrol carbon dots inhibitor
1) Resveratrol, a small molecular amyloid beta protein aggregation inhibitor, and o-phenylenediamine (oPD), a nitrogen-containing heteroatom dopant, according to a ratio of 10:40 molar concentration ratio is dissolved in deionized water, heated in a water bath kettle to be completely dissolved, then 10mL of solution is transferred into a 25mL polytetrafluoroethylene reaction kettle, and the solution is placed into an electrothermal blowing drying box to react for 12 hours at 120 ℃ by a hydrothermal method to complete the reaction;
2) And (2) putting the reaction solution in the step (1) into a dialysis bag with a cut-off molecular weight of 3000Da, dialyzing in deionized water for 24 hours, then putting into 90% ethanol for dialyzing for 24 hours, replacing the dialysate every 6 hours, removing redundant reactants and impurities, and concentrating the obtained solution by a rotary evaporation method and a freeze drying method at 50 ℃ to obtain resveratrol carbon dot powder serving as an inhibitor.
Example 6: preparation process of rosmarinic acid carbon dots inhibitor
1) Rosmarinic acid, a small molecule amyloid beta protein aggregation inhibitor, and cysteine (Cys), a sulfur-containing heteroatom dopant, were mixed according to a ratio of 2: dissolving the solution with the molar concentration ratio of 100 in deionized water, heating in a water bath kettle to dissolve completely, transferring 10mL of the solution into a 25mL polytetrafluoroethylene reaction kettle, placing into an electrothermal blowing drying box, and reacting for 6 hours at 80 ℃ by a hydrothermal method to complete the reaction;
2) And (2) putting the reaction solution in the step (1) into a dialysis bag with a molecular weight cut-off of 5000Da, dialyzing in deionized water for 72 hours, then putting into 60% ethanol for dialyzing for 72 hours, replacing the dialysate every 4 hours, removing redundant reactants and impurities, and concentrating the obtained solution by a rotary evaporation method and a freeze drying method at 55 ℃ to obtain the rosmarinic acid carbon dot inhibitor powder.
Example 7: preparation process of inhibitor Brazilian carbon spot
1) The small molecule amyloid beta protein aggregation inhibitor brazilin and the nitrogen heteroatom dopant o-phenylenediamine (oPD) were mixed according to 120: dissolving the 10 molar concentration ratio in deionized water, heating in a water bath kettle to dissolve completely, transferring 20mL of solution into a 50mL polytetrafluoroethylene reaction kettle, placing into an electrothermal blowing drying box, and reacting for 4 hours at 100 ℃ by a hydrothermal method to complete the reaction;
2) And (2) putting the reaction solution in the step (1) into a dialysis bag with the molecular weight cut-off of 100Da, dialyzing in deionized water for 12 hours, then putting into 75% ethanol for dialyzing for 12 hours, changing the dialysate every 6 hours, removing redundant reactants and impurities, and concentrating the obtained solution by a rotary evaporation method and a freeze drying method at 50 ℃ to obtain the inhibitor Brazilian carbon dot powder.
The EGCG carbon dots of example 1 were used as performance tests below. Other inhibitor carbon sites can achieve similar properties and are not described one by one.
Example 8: different concentrations of 75E-PCDs and Abeta 40 Kinetic changes in ThT fluorescence upon co-cultivation.
Aβ with a purity of 95% 40 Dissolving in Hexafluoroisopropanol (HFIP) at a concentration of 1.0mg/mL, breaking the formed aggregate by ice bath ultrasound for 30min, standing at 4deg.C for 2h to dissolve completely, centrifuging at 4deg.C for 20min at 16000g, and freezing 75% of the supernatant in a refrigerator at-70deg.C overnight. Finally put into a freeze dryer to add Aβ 40 Freeze-drying to obtain cotton flocculus, and storing in a refrigerator at-20deg.C.
Freeze-dried Abeta 40 Dissolving in 20mM NaOH solution, and performing ice bath ultrasonic treatment for 15min to obtain 275 μm Aβ 40 Mother liquor. Dilution with HEPES buffer (20 mM, pH 7.4, containing 100mM NaCl) containing 27.5. Mu.M THT gave a final concentration of 25. Mu.M Abeta 40 Solutions were used as control experiments.
0.0275-0.88mg of E-CPDs were weighed and dissolved in 10mL of HEPES buffer (20 mM, pH 7.4, containing 100mM NaCl) containing 27.5. Mu.M thT, respectively, to obtain 0.00275-0.088mg/mL of inhibitor solution. Aβ at 275. Mu.M was taken 40 The mother liquor was diluted with 75E-PCDs solution, respectively, to obtain Abeta with a final concentration of 25. Mu.M 40 A solution. 200 mu L of the solution is dripped into a 96-well plate, each group is parallel four times, and the solution is continuously measured by an enzyme-labeled instrument, the excitation wavelength is 440nm, the emission wavelength is 480nm, and the excitation light and the emission are carried outThe light emission bandwidths were all 5nm, measured at 10 minute intervals, rocked for 5s before each measurement, the temperature was set to 37℃and the fluorescence intensity at 480nm was plotted as a function of time. The results are shown in FIG. 3.
As can be seen from FIG. 3, different concentrations of 75E-PCDs and Abeta 40 When co-cultured, the concentration-dependent decrease of the ThT fluorescence intensity and the almost complete inhibition of Abeta by 80. Mu.g/mL of 75E-PCDs 40 Fiber can inhibit about 80% of Abeta at a low concentration of 2.5 μg/mL of 75E-PCDs 40 Fiber, description of Aβ 40 The beta-sheet structure has a strong inhibition effect.
Example 9: different concentrations of 75E-PCDs and Abeta 40 Circular dichroism spectrum of the culture after 72h co-culture.
Aβ was configured as in example 1 40 The mother liquor was diluted with PBS buffer (100mM PB,10mM NaCl,pH 7.4) to give A.beta.with a final concentration of 25. Mu.M 40 Solutions were used as control experiments.
Different amounts of 75E-PCDs were weighed out and dissolved in PBS buffer (100mM PB,10mM NaCl,pH 7.4) to give inhibitor solutions at concentrations of 2.75,5.5,11,22,44 and 88. Mu.g/mL, respectively. Aβ with concentration of 275. Mu.M was taken 40 The mother liquor is diluted by inhibitor solutions with different concentrations respectively to obtain the inhibitor with the final concentration of 2.5,5,10,20,40 and 80 mu g/mL and the Abeta with the final concentration of 25 mu M 40 A solution. These solutions of different concentrations were incubated with the control solution at 37℃for 72h at 150 rpm.
Taking 300 mu L of a sample with the culture time of 72 hours, adding the sample into a quartz dish with the optical path of 0.1cm, scanning a circular dichroism spectrum at the wavelength of 190-260nm, wherein the bandwidth is 1nm, the response time is 1s, the scanning speed is 100nm/min, and continuously scanning three times to obtain an average value. The intensity of the circular dichroism signal of the different samples in the wavelength range of 190-260nm is plotted. As shown in fig. 4.
As can be seen from FIG. 4, various concentrations of 75E-PCDs can effectively inhibit Abeta in a concentration-dependent relationship 40 The generation of parallel beta-sheet conformation reduces beta-sheet content, and can lead Abeta at high concentration of 80 mug/mL 40 The random coil structure is maintained.
Example 10:75E-PCDs vs. Abeta 40 Influence of aggregation morphology.
Aβ was configured as in example 1 40 The mother liquor was diluted with HEPES buffer (20 mM, pH 7.4, containing 100mM NaCl) to give a final concentration of 25. Mu.M of Abeta 40 A solution. Then respectively preparing Aβ containing 2.5-80 mug/mL 75E-PCDs 40 Solution of Abeta 40 The final concentration of (2) was 25. Mu.M. The above solution was cultured at 37℃and 150 rpm. After 120h of culture, 50. Mu.L of the mixture was dropped on mica flakes, and the mixture was allowed to stand for 5min to allow the sample to be sufficiently bonded to the mica surface. And then washing with deionized water slowly for 5 times, removing salt ions in the buffer solution, standing and airing. Observations were made with a tapping mode of an atomic force microscope (CSPM 5500, primitive). As shown in fig. 5.
As can be seen from FIG. 5A, abeta 40 After 120h of culture alone, dense fibrous aggregates were formed, and 75E-PCDs in FIG. 5B were able to effectively scavenge Abeta 40 Fibrous aggregates, in which the fibers almost completely disappeared at high concentrations, also better inhibited fiber formation at low concentrations, formed small amounts of amorphous aggregates, indicating that 75E-PCDs can significantly reduce Abeta 40 Fiber production and change of Abeta 40 Is a group of the aggregation forms of (a).
Example 11:75E-PCDs vs. Abeta 40 Alleviation of induced cytotoxicity
Aβ was configured as in example 1 40 The mother liquor was diluted with HEPES buffer (20 mM, pH 7.4, containing 100mM NaCl) to give a final concentration of 25. Mu.M of Abeta 40 A solution. Then respectively preparing Aβ containing 2.5-80 mug/mL 75E-PCDs 40 Solution of Abeta 40 The final concentration of (2) was 25. Mu.M. The above solution was incubated at 37℃for 48h at 150 rpm.
DMEM/F12 medium was prepared to which 10% fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin were added. In a sterile 96-well plate, 80. Mu.L of SH-SY5Y cells (about 8000 cells/well) were added to each well, followed by 5% CO at 37 ℃ 2 Sterile CO of (a) 2 Culturing in an incubator for 24 hours. Then, 20. Mu.L of the cultured sample and 75E-PCDs with different concentrations were added to each well, and after culturing was continued in an incubator for 24 hours, each wellmu.L of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (3 (-4, 5-dimethyl-2-thiazolyl) -2,5-diphenyl-2-H-tetrazolium bromide, MTT) solution at a concentration of 5.5mg/mL was added, and the culture was continued for 4 hours. After centrifugation of the 96-well plate (rotation speed 1500 r/min) for 10min, the medium was removed, and then 100. Mu.L of dimethyl sulfoxide (dimethyl sulfoxide, DMSO) was added to each well to lyse the cells, and the cells were shaken in an air shaker at 37℃for 20min at 150rpm to ensure complete release of blue-violet crystalline formazan, and the absorbance at 570nm was determined using a microplate reader (Tecan SUNRISE, diken). Each set of samples was added to 5 duplicate wells and their average was taken. Wells with medium alone and without cells were used as blanks, and wells with cells and HEPES buffer were used as controls. Absorbance at 570nm was plotted for different concentrations of inhibitor. As shown in fig. 6.
As can be seen from FIG. 6A, 75E-PCDs have good biocompatibility, and as can be seen from FIG. 6B, 75E-PCDs release Abeta in a concentration-dependent manner 40 Induced cytotoxicity, saving cell viability to more than 80%.
Example 12:75E-PCDs clear amyloid plaques in CL 2006.
Firstly preparing an NMG culture medium, weighing 17g of agar powder, 2.5g of peptone and 3g of sodium chloride, adding into a conical flask, adding deionized water to 1L, sterilizing at 121 ℃ for 20 minutes at high temperature, cooling to 70 ℃, sequentially adding 25mL of 1M potassium dihydrogen phosphate, 1mL of 1M magnesium sulfate, 1mL of 5mg/mL of cholesterol and 1mL of 1M calcium chloride into the conical flask, shaking uniformly, introducing into a flat plate, and airing for later use.
Preparing an LB liquid culture medium, weighing 2g of peptone, 1g of yeast powder and 2g of sodium chloride, adding into a conical flask, adding deionized water to 200mL, picking up escherichia coli OP50 to LB liquid culture medium, and culturing in a shaking table at 37 ℃ and 220rpm for 12h. And (3) coating the cultured OP50 bacterial liquid into an NGM culture medium, dripping 200 mu L of each flat plate, airing the bacterial liquid, and inversely placing the bacterial liquid into a refrigerator at 4 ℃ for standby.
200. Mu.L of 75. Mu.g/mL of E-PCDs were added to the NGM medium with OP50, and after the liquid had dried, 10L 4-stage AD model C.elegans mutant CL2006 was picked up in the dish and a corresponding 75E-PCDs-free blank was placed. After 3 days of culture, nematodes in the dishes were rinsed with 4% tissue cell fixative, fixed at 4℃for 24h, and stained with 10. Mu.M ThT solution for 4h. The stained nematodes were placed on a glass slide and observed using an inverted fluorescence microscope (TE 2000-U, nikon, japan). As shown in fig. 7.
As can be seen from fig. 7A, a distinct fluorescent spot appears in mutant CL2006, indicating the generation of a large number of amyloid plaques. When 80 μg/mL of 75E-PCDs were added in FIG. 7B, the large amount of amyloid plaques in CL2006 disappeared, indicating that 75E-PCDs were able to inhibit the formation of amyloid plaques in CL 2006.
Example 13: effect of 75E-PCDs on CL2006 survival.
An NGM medium with OP50 was prepared as in example 5, 200. Mu.L of 80. Mu.g/mL 75E-PCDs were inoculated into the medium, and a corresponding 75E-PCDs-free blank was set. To the medium, 300. Mu.L of 150. Mu.M 5-fluoro-2' -deoxyuridine was added, respectively, to suppress nematode oviposition. After the liquid was dried, 60 CL2006 nematodes at stage L4 were picked up separately into the petri dishes, and the number of nematodes surviving in the petri dishes was recorded daily until all nematodes died. To ensure adequate food supply, the transfer plate was performed every 3 days. As shown in fig. 8.
As can be seen from FIG. 8, the survival period of the individual culture of the mutant CL2006 is about 14 days, when 80 mug/mL of 75E-PCDs are added, the death rate of the CL2006 is obviously reduced from the initial stage of the culture, and the nematodes are not all killed until 20 days, which indicates that the 75E-PCDs can reduce the paralysis rate of the CL2006, clear the dyskinesia of the CL2006 and inhibit the Abeta expressed in the CL2006 body 42 Aggregation-induced cytotoxicity, and thus prolonged the lifetime of CL2006 by 6 days.
Various experimental means prove that 75E-PCDs can obviously inhibit Abeta at low concentration 40 Is effective in relieving Abeta 40 Induced cytotoxicity, clearing amyloid deposits in AD model caenorhabditis elegans CL2006, and can prolong the survival of CL 2006. Inhibition of Abeta by 75E-PCDs 40 The aggregation ability is achieved by the ThT kinetic experiment and atomic force microscopyThe method is used for relieving Abeta by measuring through a mirror experiment, a circular dichroism spectrum experiment and the like 40 The ability to induce cytotoxicity was demonstrated by 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT) assay experiments, whose ability to clear amyloid deposits in CL2006 and extend CL2006 survival was determined by fluorescence microscopy experiments and life cycle experiments.
The invention innovatively provides a design of taking a small molecular amyloid beta protein inhibitor as a main carbon source to obtain an inhibitor carbon point, provides application of the inhibitor carbon point in the aspects of preparing medicines for inhibiting amyloid beta protein aggregation and the like, and applies the inhibitor carbon point to Abeta 40 Conformational change, aggregation and cytotoxicity inhibition experiments. While the present preferred embodiments have been described in the field, it will be apparent to those skilled in the relevant art that modifications and appropriate changes and combinations of the methods described herein can be made to practice the present technology without departing from the spirit and scope of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be included within the spirit, scope and content of the invention.

Claims (2)

1. The application of carbon point inhibitor synthesized by using EGCG as raw material for inhibiting aggregation of amyloid beta protein in preparing medicine for treating Alzheimer's disease of aggregation of amyloid beta protein in brain of patient is characterized by that it utilizes carbon point synthesized by EGCG, EGCG possesses good inhibiting action for aggregation of amyloid beta protein, makes EGCG as main carbon source and doped with o-phenylenediamine, and utilizes one-step hydrothermal method to obtain carbon point inhibitor synthesized by using EGCG as raw material, and can effectively inhibit A beta at low concentration 40 Is effective in relieving Abeta 40 Induced cytotoxicity, clearing amyloid plaques in the body of caenorhabditis elegans CL2006 of the AD model and prolonging nematode life, can alleviate AD symptoms.
2. Use of a carbon point inhibitor synthesized from EGCG as a raw material for inhibiting the aggregation of amyloid β protein as claimed in claim 1 for the manufacture of a medicament for the treatment of alzheimer's disease in which amyloid β protein aggregates in the brain of a patient, wherein the method for the manufacture of the carbon point inhibitor comprises the steps of:
1) EGCG and o-phenylenediamine were prepared according to 75:50 molar concentration ratio is dissolved in deionized water, and the reaction is completed at 200 ℃ through a hydrothermal method;
2) Dialyzing the reaction solution in the step 1) in deionized water for 48 hours, then dialyzing the reaction solution in 50% ethanol for 48 hours, and concentrating the obtained solution by a spin-steaming method and a freeze-drying method to obtain carbon dot powder synthesized by using EGCG as a raw material.
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