CN114377033A - Preparation method and application of inhibitor carbon dots for inhibiting amyloid beta protein aggregation - Google Patents

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

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CN114377033A
CN114377033A CN202210180468.8A CN202210180468A CN114377033A CN 114377033 A CN114377033 A CN 114377033A CN 202210180468 A CN202210180468 A CN 202210180468A CN 114377033 A CN114377033 A CN 114377033A
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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 small molecular amyloid beta protein aggregation inhibitor and a dopant containing heteroatoms in deionized water, heating the mixture in a water bath kettle to completely dissolve the small molecular amyloid beta protein aggregation inhibitor and the dopant, transferring the solution with the volume not exceeding 2/3 of the reaction kettle into a polytetrafluoroethylene reaction kettle, putting the polytetrafluoroethylene reaction kettle into an electrothermal blowing dry box, and completing the reaction by a hydrothermal method; putting the reaction solution into a dialysis bag with molecular weight cutoff for dialysis, removing redundant reactants and impurities, and concentrating the obtained solution by a rotary evaporation method and a freeze drying method to obtain the inhibitor carbon dot powder. The carbon dots of the inhibitor can effectively inhibit A beta at low concentration40Significantly reduce A beta40And (3) generation of a beta-sheet structure in the aggregation process. The inhibitor carbon dots can inhibitA beta expressed in CL2006 body42The toxicity generated by aggregation induction and the prolongation of the CL2006 life span are ideal inhibitors of amyloid beta protein aggregation.

Description

Preparation method and application of inhibitor carbon dots 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 Alzheimer's Disease have clinical symptoms such as memory loss, cognitive dysfunction, and directional loss (Lancet,2006,368: 387-. AD is the most common cause of dementia in the elderly, with approximately 5000 more than ten thousand affected in 2020, 8300 ten thousand in 2030 and 1.52 billion in 2050 (Molecules, 2021, 26 (6)). At present, The pathogenic mechanism of AD is not clearly studied, and some researchers have proposed several hypotheses on The pathogenic factors of AD, including The inflammation hypothesis, The tau hypothesis, The Amyloid-beta protein (A β) hypothesis and The cholinergic hypothesis, in which The Amyloid-beta protein cascade hypothesis (ACH) is widely accepted (Science of The Total environmental, 2020,700: 134836). ACH suggests that The accumulation of amyloid-beta peptide in The brain occurs early in AD, which then can lead to The formation of Senile Plaques (SPs) and subsequent neurofibrillary tangles (NFTs), causing neuronal cell death, and ultimately dementia (The Protein Journal, 2019,38(4): 425-434). Amyloid beta Protein is a small peptide consisting of 39-43 amino acids generated by the cleavage of Amyloid Precursor Protein (APP) by beta-and gamma-secretases, the most common form of Amyloid beta Protein being 40 amino acids of A beta40And 42 amino acids of A beta42Two (The Protein Journal, 2019,38(4):425 and 434). Amyloid beta protein aggregates in the brain of a patient, self-assembles into soluble amyloid beta oligomers and insoluble amyloid beta fibers, and exerts toxic and oxidative stress on neuronal cells, which may even gradually lead to the development of synaptic dysfunction of neurons, resulting in the death of neurons, and finally in the damage of the nervous system of the patient (Science, 2002, 297(5580): 353-) -356). Therefore, an appropriate amyloid beta protein inhibitor is designed, and the interaction between the inhibitor and amyloid beta protein is utilized to block the shape of amyloid beta protein fibersThe formation of low-toxicity amorphous aggregates by routing or disrupting the beta sheet in amyloid beta fibrillar structures can alleviate AD symptoms.
The amyloid beta protein inhibitor comprises small molecule inhibitor, polypeptide inhibitor, protein inhibitor and nanoparticle inhibitor. The small molecule inhibitor has simple structure, obvious effect and good BBB penetration performance, but most small molecule inhibitors have poor water solubility and low specificity, thereby causing low bioavailability (ACS Chemical Neuroscience, 2019, 10(8): 3366-3374); the polypeptide inhibitor has the advantages of small molecular weight, easy synthesis, no immunogenicity and strong specificity, but is easy to self-aggregate, has higher effective concentration and is difficult to pass through BBB (ACS Chemical Neuroscience, 2017, 9(2): 198-210); protein inhibitors, although having good biocompatibility, are difficult to pass through BBB (ACS Chemical Neuroscience, 2019, 10(8): 3366-3374). In contrast, although the nanoparticle inhibitor has low specificity to amyloid beta protein, the nanoparticle inhibitor has a large specific surface area and is simple to synthesize, and other inhibitors (such as small molecules, peptides, proteins and the like) can be introduced through surface modification, so that the nanoparticle inhibitor is an inhibitor with wide research and development prospects. The nano materials are in a wide variety, and in recent years, Quantum Dots (QDs) having unique physicochemical properties have attracted wide attention of the scientific community due to their applications in medicine. Wherein, the carbon quantum dots (CDs) have unique structure and property, and conjugated pi electrons in the CDs can generate pi-pi stacking interaction with aromatic rings of amino acid residues in the amyloid polypeptide so as to influence an amyloid beta protein aggregation path (Adv Mater, 2013, 25 (28): 3780-801). Some studies have reported that CDs have significant inhibitory effects on amyloidosis (Nanoscale, 2019, 11(46):22387-22397) and can be used as detection probes for amyloid (Protein and Peptide Letters, 2019). In addition, CDs also 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 have little effect on inhibiting the aggregation of amyloid beta protein and most CDs have no effect on inhibiting the aggregation of amyloid beta protein.
Many organic small molecule inhibitors have good inhibitory effect on amyloid beta protein aggregation, such as Epigallocatechin gallate (EGCG), curcumin, resveratrol and brazilin, 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 use 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 found in food, and various studies have shown that natural polyphenols inhibit the formation of amyloid fibrils and exhibit good resistance to amyloidosis (Molecular differentiation & food research,2015,59: 8-20). EGCG is a green natural plant polyphenol and can obviously inhibit the formation of amyloid beta protein aggregates. In addition, The EGCG structure contains a catechol structure, and researches show that The polyphenol compound has wide biological activities including metal chelation, anti-inflammatory activity, anticancer activity, antimicrobial activity, antioxidant activity and The like (The Science of The Total Environment,2020,700:134836.1-134836.11), and The phenol compound with The catechol structure can inhibit The aggregation of amyloid beta protein and human pancreatic amyloid polypeptide (hIAPP), and simultaneously has antioxidant activity and protective effect (Natural Product Communications,2019,14(5):1934578X 1984303). Studies show that the incorporation of heteroatoms has a great influence on the properties of 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 fluorescence properties of the carbon dots; and o-phenylenediamine is used as a nitrogen source, so that amino groups can be retained on the surfaces of the carbon dots, the surface charges of the carbon dots can be improved, the positively charged amino groups on the surfaces of the carbon dots and negatively charged amyloid beta protein can generate electrostatic interaction, and the effect of inhibiting the aggregation of the amyloid beta protein is achieved (Small,2020, 16).
The invention creatively designs based on the small-molecule amyloid beta protein inhibitor, and provides a method for synthesizing carbon dots by using an organic small-molecule 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-molecule inhibitor and generate the multifunctional carbon dot inhibitor. The carbon-containing amyloid beta protein aggregation small molecule inhibitor is used as a precursor, and part of functional groups of the carbon-containing amyloid beta protein aggregation small molecule inhibitor are probably effective methods for improving the interaction between carbon points and amyloid beta protein and enhancing the carbon points to inhibit the amyloid beta protein aggregation, and are also effective strategies for adjusting CDs to play multiple functions. Therefore, the invention provides a new research visual angle for the research and development of the multifunctional high-efficiency amyloid beta protein aggregation inhibitor.
Therefore, the invention is innovatively designed based on the small-molecular amyloid beta protein inhibitor, the small-molecular amyloid beta protein aggregation inhibitor is used as a main carbon source, and the dopant containing the heteroatom is introduced to obtain inhibitor carbon dots (the carbon dots are named as E-PCDs), the E-PCDs and the amyloid beta protein have higher affinity, and the aggregation of the amyloid beta protein can be effectively inhibited under low concentration. This indicates that the carbonaceous amyloid beta protein aggregation small molecule inhibitor is used as a precursor, and a part of functional groups of the carbonaceous amyloid beta protein aggregation small molecule inhibitor is probably an effective method for improving the interaction between carbon points and amyloid beta protein and enhancing the carbon points to inhibit the aggregation of the amyloid beta protein, and is also an effective strategy for adjusting CDs to play multiple functions. Therefore, the invention provides a new research visual angle for the research and development of the multifunctional high-efficiency amyloid beta protein aggregation inhibitor.
The invention aims to provide an inhibitor carbon dot with a strong function of inhibiting amyloid beta protein aggregation, a preparation method and application thereof in inhibiting amyloid beta protein aggregation, wherein the structural expression is shown in figure 1. The carbon dots of the inhibitor have obvious inhibition effect on amyloid beta protein aggregation and can relieve A beta40Induced cytotoxicity, eliminating amyloid plaques in an AD model caenorhabditis elegans CL2006 body and prolonging the life of the nematode, and the specific technical scheme is as follows:
a carbon dot synthesized by using a small-molecular amyloid beta protein inhibitor is used, the small-molecular amyloid beta protein aggregation inhibitor is used as a main carbon source and is doped with a dopant containing hetero atoms, the inhibitor carbon dots (E-PCDs) are 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 dopant containing a heteroatom by utilizing a carbon point synthesized by an organic small-molecular amyloid beta protein inhibitor through a one-step hydrothermal method.
The preparation method of the inhibitor carbon dot for inhibiting amyloid beta protein aggregation comprises the following steps:
1) respectively mixing the small-molecule amyloid beta protein aggregation inhibitor and the dopant containing the heteroatom according to the ratio of 2-120: dissolving the mixture in deionized water according to the molar concentration ratio of 10-100, heating the mixture in a water bath kettle to completely dissolve the mixture, transferring the solution with the volume not exceeding 2/3 of the reaction kettle into a polytetrafluoroethylene reaction kettle, putting the polytetrafluoroethylene reaction kettle into an electrothermal blowing dry box, and reacting the mixture for 4-12 hours at 80-200 ℃ by a hydrothermal method to complete the reaction;
2) putting the reaction solution in the step 1) into a dialysis bag with the molecular weight cutoff of 100-.
The small molecular amyloid beta protein aggregation inhibitor comprises one of EGCG, curcumin, brazilein, resveratrol, human serum albumin or other small molecular inhibitors.
The dopant containing the heteroatom comprises o-phenylenediamine, cysteine or other dopants containing the heteroatom.
The preferable molar concentration ratio of the small molecular amyloid beta protein aggregation inhibitor to the dopant containing the heteroatom is 10-75: 40-50.
The preferred hydrothermal reaction condition is 120-200 ℃ and the reaction time is 10-12 h.
Preferably, the cut-off molecular weight of the dialysis bag is 500-3000Da, and the dialysis time is 24-48 h.
Preferably, the solution obtained by filtering and dialyzing the solution by using a 0.22 mu m nylon membrane before rotary evaporation, wherein the rotary evaporation temperature is 50-60 ℃.
The carbon dots of the inhibitor for inhibiting the aggregation of amyloid beta protein can be used for preparing the medicine for treating Alzheimer's disease.
The preferred volumes of the reaction vessel are 25 and 50mL, corresponding reaction solvent systems are 10 and 20mL deionized water, respectively.
The inhibitor carbon dot with the function of inhibiting amyloid beta protein aggregation, the preparation method and the application of the inhibitor carbon dot in inhibiting amyloid beta protein aggregation have the following advantages:
first, the inhibitor carbon dot is effective at inhibiting a β at low concentrations40Significantly reduce A beta40And (3) generation of a beta-sheet structure in the aggregation process.
Second, the inhibitor carbon site can alter A β40Inhibiting the formation of amyloid fibrils.
Thirdly, the inhibitor carbon dots have good biocompatibility and can relieve A beta40Induced cytotoxicity.
Fourthly, the carbon dots of the inhibitor can inhibit the formation of amyloid plaques in the body of the AD model C.elegans CL2006 and eliminate dyskinesia of the CL 2006.
Fifth, the inhibitor carbon dots are capable of inhibiting A β expressed in CL2006 in vivo42The toxicity generated by aggregation induction and the prolongation of the CL2006 life span are ideal inhibitors of amyloid beta protein aggregation.
The invention provides inhibitor carbon dots (E-PCDs) capable of inhibiting amyloid beta protein aggregation, wherein small-molecule amyloid beta protein aggregation inhibitors and nitrogen-containing heteroatom dopants are taken as examples in the invention, the small-molecule amyloid beta protein aggregation inhibitors are taken as main carbon sources, the nitrogen-containing heteroatom dopants are added, and the inhibitor carbon dots (E-PCDs) are synthesized by a green one-step hydrothermal method. The E-PCDs have the capability of inhibiting the aggregation of amyloid beta protein, and the E-PCDs and A beta protein with different concentrations40When co-cultured, A.beta.can be decreased in a concentration-dependent manner40ThT fluorescence intensity of (2.5. mu.g/mL) at a low concentrationE-PCDs can also inhibit A beta by about 80%40Fibers, description of E-PCDs vs. A β40Aggregation into a beta-sheet structure has a strong inhibition effect; the circular dichroism spectrum proves that the E-PCDs can effectively inhibit A beta in a concentration-dependent relationship40The generation of parallel beta-sheet conformation, the reduction of beta-sheet content and the high concentration of 80 mu g/mL can lead A beta to be40Keeping a random coil structure; AFM (atomic force microscopy) experiments show that the E-PCDs can obviously reduce Abeta40Production of fiber and modification of Abeta40(ii) an aggregated morphology; cell experiments prove that the E-PCDs have good biocompatibility and can relieve A beta40Induced 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 bodies, and the life of the CL2006 is prolonged by 6 days.
The carbon point E-PCDs of the invention are applied to the preparation of the medicines for treating Alzheimer's disease.
Drawings
FIG. 1: schematic structural diagram of E-PCDs.
FIG. 2: schematic diagram of the synthetic pathway of E-PCDs.
FIG. 3: example 8 varying concentrations of E-PCDs and A β40ThT fluorescence kinetics profiles upon co-culture.
FIG. 4: example 9 varying concentrations of E-PCDs and A β40Circular Dichroism (CD) spectrogram of culture after 72h co-culture
FIG. 5: example 10 varying concentrations of E-PCDs and A β40Topography of the culture after 120h co-cultivation.
FIG. 6: example 11 varying concentrations of E-PCDs and A β40Cell viability profile after co-culture with SH-SY5Y cells.
FIG. 7: fluorescence microscopy of E-PCDs clearing amyloid plaques in CL2006 in example 12.
FIG. 8: survival of CL2006 in example 13 after co-culture of E-PCDs with CL 2006.
Detailed Description
The present invention will be further described with reference to the following specific examples.
Example 1: preparation process of carbon point of inhibitor EGCG
1) Mixing a small-molecule amyloid beta protein aggregation inhibitor EGCG and a nitrogen-containing heteroatom dopant o-phenylenediamine (oPD) according to the ratio of 75: dissolving 50 mol concentration ratio in deionized water, heating in a water bath to completely dissolve, transferring 10mL of solution into a 25mL polytetrafluoroethylene reaction kettle, putting into an electrothermal blowing dry box, and reacting at 200 ℃ for 10h by a hydrothermal method to complete the reaction;
2) putting the reaction liquid obtained in the step 1) into a dialysis bag with the molecular weight cutoff of 500Da, dialyzing in deionized water for 48h, then putting into 50% ethanol for dialyzing for 48h, replacing the dialyzate every 6h, removing redundant reactants and impurities, concentrating the obtained solution by a rotary evaporation method at 55 ℃ and a freeze-drying method, and obtaining the EGCG carbon dot (named as 75E-PCDs) powder serving as an inhibitor.
Example 2:
1) mixing a small-molecule amyloid beta protein aggregation inhibitor EGCG and a nitrogen-containing heteroatom dopant o-phenylenediamine (oPD) according to the ratio of 10: dissolving 50 mol concentration ratio in deionized water, heating in a water bath to completely dissolve, transferring 10mL of solution into a 25mL polytetrafluoroethylene reaction kettle, putting into an electrothermal blowing dry box, and reacting at 120 ℃ for 10 hours by a hydrothermal method to complete the reaction;
2) putting the reaction liquid obtained in the step 1) into a dialysis bag with the molecular weight cutoff of 100Da, dialyzing in deionized water for 24h, then putting into 50% ethanol for dialysis for 24h, replacing the dialysis liquid every 6h, removing redundant reactants and impurities, concentrating the obtained solution by a rotary evaporation method at 55 ℃ and a freeze-drying method, and obtaining the powder of the EGCG carbon dots (named as 10E-PCDs) serving as the inhibitor.
Example 3:
1) mixing a small-molecule amyloid beta protein aggregation inhibitor EGCG and a nitrogen-containing heteroatom dopant o-phenylenediamine (oPD) according to the ratio of 120: dissolving 50 mol concentration ratio in deionized water, heating in a water bath to completely dissolve, transferring 20mL of solution into a 50mL polytetrafluoroethylene reaction kettle, putting into an electrothermal blowing dry box, and reacting at 180 ℃ for 12h by a hydrothermal method to complete the reaction;
2) putting the reaction liquid obtained in the step 1) into a dialysis bag with the molecular weight cutoff of 1000Da, dialyzing in deionized water for 48h, then putting into 50% ethanol for dialyzing for 48h, replacing the dialyzate every 6h, removing redundant reactants and impurities, concentrating the obtained solution by a rotary evaporation method at 55 ℃ and a freeze-drying method, and obtaining the EGCG carbon dot (named as 120E-PCDs) powder serving as an inhibitor.
Example 4: process for preparing curcumin carbon dots inhibitor
1) Curcumin, a small-molecule amyloid beta protein aggregation inhibitor, and cysteine (Cys), a sulfur-containing heteroatom dopant, were mixed according to a 40: dissolving 45 mol% of the solution in deionized water, heating the solution in a water bath kettle to completely dissolve the solution, transferring 20mL of the solution into a 50mL polytetrafluoroethylene reaction kettle, placing the reaction kettle into an electrothermal blowing dry box, and reacting the solution for 11 hours at 150 ℃ by a hydrothermal method to complete the reaction;
2) putting the reaction solution obtained in the step 1) into a dialysis bag with the molecular weight cutoff of 1500Da, dialyzing in deionized water for 36h, then dialyzing in absolute ethyl alcohol for 36h, replacing the dialysis solution every 5h, removing redundant reactants and impurities, and concentrating the obtained solution by a 60 ℃ rotary evaporation method and a freeze drying method to obtain the inhibitor curcumin carbon dot powder.
Example 5: preparation process of inhibitor resveratrol carbon dots
1) Resveratrol, a small-molecule amyloid beta protein aggregation inhibitor, and o-phenylenediamine (oPD), a nitrogen-containing heteroatom dopant, were added in a ratio of 10: dissolving 40 mol% of the solution in deionized water, heating the solution in a water bath kettle to completely dissolve the solution, transferring 10mL of the solution into a 25mL polytetrafluoroethylene reaction kettle, putting the reaction kettle into an electrothermal blowing dry box, and reacting for 12 hours at 120 ℃ by a hydrothermal method to complete the reaction;
2) putting the reaction solution obtained in the step 1) into a dialysis bag with the molecular weight cutoff of 3000Da, dialyzing in deionized water for 24h, then putting into 90% ethanol for dialysis for 24h, replacing the dialysis solution every 6h, removing redundant reactants and impurities, and concentrating the obtained solution by a 50 ℃ rotary evaporation method and a freeze drying method to obtain the inhibitor resveratrol carbon dot powder.
Example 6: preparation process of inhibitor rosmarinic acid carbon dots
1) Rosmarinic acid, a small-molecule amyloid beta protein aggregation inhibitor, and cysteine (Cys), a sulfur-containing heteroatom dopant, were added according to a 2: dissolving 100 mol concentration ratio in deionized water, heating in a water bath kettle for complete dissolution, transferring 10mL of solution into a 25mL polytetrafluoroethylene reaction kettle, placing the reaction kettle into an electrothermal blowing dry box, and reacting for 6 hours at 80 ℃ by a hydrothermal method to complete the reaction;
2) putting the reaction solution obtained in the step 1) into a dialysis bag with the molecular weight cutoff of 5000Da, dialyzing in deionized water for 72h, then putting the dialysis bag into 60% ethanol for dialysis for 72h, replacing the dialysis solution every 4h, removing redundant reactants and impurities, and concentrating the obtained solution by a rotary evaporation method at 55 ℃ and a freeze drying method to obtain the inhibitor rosmarinic acid carbon dot powder.
Example 7: preparation process of inhibitor brazilin carbon dots
1) Mixing small-molecule amyloid beta protein aggregation inhibitor brazilein and nitrogen-containing heteroatom dopant o-phenylenediamine (oPD) according to the ratio of 120: dissolving the 10 molar concentration ratio in deionized water, heating the mixture in a water bath kettle to completely dissolve the mixture, transferring 20mL of solution into a 50mL polytetrafluoroethylene reaction kettle, putting the reaction kettle into an electrothermal blowing dry box, and reacting the mixture for 4 hours at 100 ℃ by a hydrothermal method to complete the reaction;
2) putting the reaction solution obtained in the step 1) into a dialysis bag with the molecular weight cutoff of 100Da, dialyzing in deionized water for 12h, then putting into 75% ethanol for dialysis for 12h, replacing the dialysis solution every 6h, removing redundant reactants and impurities, and concentrating the obtained solution by a rotary evaporation method at 50 ℃ and a freeze-drying method to obtain the inhibitor Brazilian lignin carbon dot powder.
The EGCG carbon sites of example 1 were used as performance tests below. Other inhibitor carbon sites can achieve similar performance, not to mention.
Example 8: different concentrations of 75E-PCDs and Abeta40Kinetic changes in ThT fluorescence upon co-culture.
Mixing A beta with the purity of 95 percent40Dissolving in Hexafluoroisopropanol (HFIP) at a concentration of 1.0mg/mL, and ultrasonic disrupting the solution for 30min in ice bathStanding the formed aggregate at 4 deg.C for 2 hr to dissolve it sufficiently, centrifuging at 4 deg.C and 16000g for 20min, and freezing 75% of the supernatant in-70 deg.C refrigerator overnight. Finally, the A beta is put into a freeze dryer to be dried40Freeze-drying into cotton-like powder, and storing in a refrigerator at-20 deg.C.
Freeze-drying Abeta40Dissolving in 20mM NaOH solution, and ultrasonic treating in ice bath for 15min to obtain 275 μ M Abeta40And (4) mother liquor. Diluted with HEPES buffer (20mM, pH 7.4, containing 100mM NaCl) containing 27.5. mu.M ThT to give A.beta.at a final concentration of 25. mu.M40Solution, as a control experiment.
0.0275-0.88mg of E-CPDs were weighed out and dissolved in 10mL of HEPES buffer (20mM, pH 7.4, containing 100mM NaCl) containing 27.5. mu.M ThT, respectively, to obtain 0.00275-0.088mg/mL of inhibitor solution. Take 275. mu.M of Abeta40The mother liquor was diluted with 75E-PCDs solution to obtain Abeta at a final concentration of 25. mu.M40And (3) solution. 200 mu L of the fluorescent powder is dripped into a 96-well plate, each group is divided into four times of parallel measurement, continuous measurement is carried out by an enzyme-linked immunosorbent assay, the excitation wavelength is 440nm, the emission wavelength is 480nm, the bandwidth of the excitation light and the emission light is 5nm, the measurement is carried out once every 10 minutes, the temperature is set to 37 ℃ before each measurement, and the change of the fluorescence intensity at 480nm along with the time is plotted. The results are shown in FIG. 3.
As can be seen from FIG. 3, the different concentrations of 75E-PCDs and A β40When co-cultured, ThT fluorescence intensity can be reduced in a concentration-dependent manner, and 80. mu.g/mL of 75E-PCDs can almost completely inhibit A.beta.40The fiber can inhibit about 80 percent of A beta under the condition of low concentration of 2.5 mu g/mL of 75E-PCDs40Fibers, description of A.beta.40Aggregation into a beta-sheet structure has a strong inhibition effect.
Example 9: different concentrations of 75E-PCDs and Abeta40Circular dichroism spectrum of the culture after 72h of co-cultivation.
Ass preparation by the method of example 140The mother liquor was diluted with PBS buffer (100mM PB, 10mM NaCl, pH 7.4) to give A.beta.at a final concentration of 25. mu.M40Solution, as a control experiment.
Weighing 75E-PCDs with different massesThe inhibitor solutions were dissolved in PBS buffer (100mM PB, 10mM NaCl, pH 7.4) to give concentrations of 2.75,5.5,11,22,44 and 88. mu.g/mL, respectively. Taking Abeta with the concentration of 275 mu M40The mother liquor was diluted with inhibitor solutions of different concentrations to give final concentrations of 2.5,5,10,20,40 and 80. mu.g/mL inhibitor and 25. mu.M A.beta.40And (3) solution. These solutions of different concentrations were incubated for 72h at 37 ℃ and 150rpm together with the control solution.
And adding a sample with the culture time of 72h of 300 mu L into a quartz dish with the optical path of 0.1cm, scanning the circular dichroism spectrum under the wavelength of 190 plus 260nm, wherein the bandwidth is 1nm, the response time is 1s, the scanning speed is 100nm/min, and continuously scanning for three times to obtain an average value. The intensity of the circular dichroism signal in the wavelength range of 190-260nm for the different samples was plotted. As shown in fig. 4.
As can be seen from FIG. 4, different concentrations of 75E-PCDs can effectively inhibit A beta in a concentration-dependent manner40The generation of parallel beta-sheet conformation, the reduction of beta-sheet content and the high concentration of 80 mu g/mL can lead A beta to be40The random coil structure is maintained.
Example 10: 75E-PCDs vs. Abeta40Influence of aggregation morphology.
Ass preparation by the method of example 140The mother liquor was diluted with HEPES buffer (20mM, pH 7.4, containing 100mM NaCl) to give A.beta.at a final concentration of 25. mu.M40And (3) solution. Respectively preparing A beta containing 75E-PCDs at 2.5-80 mu g/mL40Solution of A beta40The final concentration of (3) was 25. mu.M. The above solution was incubated at 37 ℃ and 150 rpm. After 120h of incubation, 50. mu.L of the solution was dropped on a mica plate and allowed to stand for 5min to allow the sample to be sufficiently bound to the mica surface. And then slowly washing with deionized water for 5 times, removing salt ions in the buffer solution, and standing and airing. The observation was carried out in the tapping mode of an atomic force microscope (CSPM5500, original). As shown in fig. 5.
As can be seen in FIG. 5A, A β40After 120h of single culture, dense fibrous aggregates are formed, and 75E-PCDs in FIG. 5B can effectively eliminate A beta40Fibrous aggregates which almost completely disappear at high concentrations and which can be well inhibited even at low concentrationsDimension generation, forming small amount of amorphous aggregates, indicating that 75E-PCDs can significantly reduce A beta40Production of fiber and modification of Abeta40The aggregate form of (1).
Example 11: 75E-PCDs vs. Abeta40Alleviation of induced cytotoxicity
Ass preparation by the method of example 140The mother liquor was diluted with HEPES buffer (20mM, pH 7.4, containing 100mM NaCl) to give A.beta.at a final concentration of 25. mu.M40And (3) solution. Respectively preparing A beta containing 75E-PCDs at 2.5-80 mu g/mL40Solution of A beta40The final concentration of (3) was 25. mu.M. The solution was incubated at 37 ℃ and 150rpm for 48 h.
DMEM/F12 medium was prepared with 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin. In a sterile 96-well plate, 80. mu.L of SH-SY5Y cells (about 8000 cells/well) were added per well, and then 5% CO was contained at 37 ℃2Sterile CO of2Culturing in an incubator for 24 h. 20 mul of the sample after 48H of culture and 75E-PCDs with different concentrations are added into each well, after the culture is continued for 24H in the incubator, 10 mul 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 with the concentration of 5.5mg/mL is added into each well, and the culture is continued for 4H. After centrifugation (1500 r/min) of 96-well plates for 10min, the medium was removed, and then 100. mu.L of dimethyl sulfoxide (DMSO) was added to each well to lyse the cells, and the cells were shaken at 150rpm in an air shaker at 37 ℃ for 20min to ensure complete release of blue-violet crystalline formazan, and the cell activity was determined by measuring the absorbance at 570nm using a microplate reader (Tecan SUNRISE, Tekken). Each set of samples was added to 5 duplicate wells and averaged. Wells with medium only and no cells added were used as blanks, and wells with cells and HEPES buffer were used as controls. The absorbance at 570nm for different concentrations of inhibitor was plotted. 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 provide a concentration-dependent alleviation of A.beta.40Induced cytotoxicity, saving cell viability to more than 80%.
Example 12: clearance of amyloid plaques in CL2006 by 75E-PCDs.
Firstly, preparing an NMG culture medium, weighing 17g of agar powder, 2.5g of peptone and 3g of sodium chloride, adding the agar powder, the peptone and the sodium chloride into a conical flask, adding deionized water to 1L, sterilizing at the high temperature of 121 ℃ for 20 minutes, cooling to 70 ℃, adding 25mL of 1M potassium dihydrogen phosphate, 1mL of 1M magnesium sulfate, 1mL of 5mg/mL cholesterol and 1mL of 1M calcium chloride into the conical flask in sequence, 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, selecting escherichia coli OP50 to the LB liquid culture medium, and culturing in a shaking table for 12h at 37 ℃ and 220 rpm. And (3) coating the cultured OP50 bacterial liquid into an NGM culture medium, dripping 200 mu L of bacterial liquid into each plate, and inversely placing the plates into a refrigerator at 4 ℃ for later use after the bacterial liquid is dried.
200 μ L of 75E-PCDs 80 μ g/mL were added to NGM medium with OP50, and after the liquid was dried, 10L 4 mutant forms of the AD model C.elegans mutant CL2006 were picked up in petri dishes and a corresponding blank control without 75E-PCDs was set. After 3 days of culture, the nematodes were washed out of the dishes using 4% tissue cell fixative, fixed at 4 ℃ for 24h, and stained with 10 μ M ThT solution for 4 h. The stained nematodes were placed on a glass slide and observed using an inverted fluorescence microscope (TE2000-U, Nikon, Japan). As shown in fig. 7.
As can be seen in fig. 7A, distinct fluorescent spots appear in mutant CL2006, indicating the generation of a large number of amyloid plaques. FIG. 7B shows that when 80. mu.g/mL of 75E-PCDs are added, a large number of amyloid plaques in CL2006 disappear, indicating that the 75E-PCDs can inhibit the formation of amyloid plaques in CL 2006.
Example 13: effect of 75E-PCDs on survival of CL 2006.
NGM medium with OP50 was prepared according to the method of example 5, 200. mu.L of 75E-PCDs 80. mu.g/mL were inoculated into the medium, and a corresponding blank control group without 75E-PCDs was set. Addition of 300. mu.L of 150. mu.M each of 5-fluoro-2' -deoxyuridine to the medium inhibited nematode oviposition. After the liquid was dried, 60L 4 stage CL2006 nematodes were picked up into the dishes, and the number of nematodes surviving in the dishes was recorded daily until all nematodes died. To ensure adequate food supply, the plate was rotated every 3 days. As shown in fig. 8.
As can be seen from FIG. 8, the survival cycle of the mutant CL2006 cultured alone is about 14 days, when 80. mu.g/mL of 75E-PCDs are added, the mortality rate of CL2006 is obviously reduced from the initial stage of culture, and nematodes die not all until the 20 th day, which shows that the 75E-PCDs can reduce the paralysis rate of CL2006, eliminate the dyskinesia of CL2006 and inhibit the A beta expressed in CL2006 body42Aggregation-induced cytotoxicity, which in turn extended the lifespan of CL2006 for 6 days.
A plurality of experimental means prove that 75E-PCDs can obviously inhibit Abeta under low concentration40Can relieve Abeta40Induced cytotoxicity, clearance of amyloid deposits in the AD model caenorhabditis elegans CL2006, and ability to prolong the survival of CL 2006. 75E-PCDs inhibit A beta40The ability to aggregate is determined by methods such as ThT kinetic experiments, atomic force microscopy experiments, and circular dichroism spectroscopy experiments, which mitigate A β40The ability to induce cytotoxicity was demonstrated by 3- (4, 5-dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide (MTT) assay experiments, and its ability to clear amyloid deposits in CL2006 and prolong CL2006 survival was determined by fluorescence microscopy experiments and life cycle experiments.
The invention innovatively provides a design for obtaining an inhibitor carbon point by taking a small-molecular amyloid beta protein inhibitor as a main carbon source, 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 A beta protein40Conformational change, aggregation and cytotoxicity inhibition assays of (1). Having described preferred embodiments in the field, it will be apparent to those skilled in the art that the techniques of the present invention may be practiced with modification, or with appropriate modification and combination, of the methods described herein without departing from the spirit, scope, and spirit of the invention. It is expressly intended that all such similar substitutes and modifications will be apparent to those skilled in the art,they are deemed to be within the spirit, scope and content of the invention.

Claims (9)

1. The inhibitor carbon dot for inhibiting amyloid beta protein aggregation is characterized in that the inhibitor carbon dot is obtained by a one-step hydrothermal method by using a carbon dot synthesized by an organic small-molecule amyloid beta protein inhibitor and taking the small-molecule amyloid beta protein aggregation inhibitor as a main carbon source and doping a dopant containing a heteroatom.
2. The preparation method of the inhibitor carbon dot for inhibiting the amyloid beta protein aggregation is characterized by comprising the following steps:
1) mixing small-molecule amyloid beta protein aggregation inhibitor and dopant containing heteroatom according to the ratio of 2-120: dissolving the mixture in deionized water according to the molar concentration ratio of 10-100, heating the mixture in a water bath kettle to completely dissolve the mixture, transferring the solution with the volume not exceeding 2/3 of the reaction kettle into a polytetrafluoroethylene reaction kettle, putting the polytetrafluoroethylene reaction kettle into an electrothermal blowing dry box, and reacting the mixture for 4-12 hours at 80-200 ℃ by a hydrothermal method to complete the reaction;
2) putting the reaction solution in the step 1) into a dialysis bag with the molecular weight cutoff of 100-.
3. The method according to claim 2, wherein the small-molecule amyloid beta protein aggregation inhibitor comprises one of EGCG, curcumin, brazilin, resveratrol, human serum albumin or other small-molecule inhibitors.
4. The method of claim 2, wherein the heteroatom-containing dopant comprises o-phenylenediamine, cysteine, or other heteroatom-containing dopants.
5. The method according to claim 2, wherein the small molecule amyloid beta aggregation inhibitor and the heteroatom-containing dopant are preferably present in a molar concentration ratio of 10 to 75: 40-50.
6. The process as claimed in claim 2, wherein the hydrothermal reaction conditions are preferably 120 ℃ to 200 ℃ and the reaction time is 10-12 h.
7. The method as claimed in claim 2, wherein the dialysis bag preferably has a molecular weight cut-off of 500-.
8. The process as claimed in claim 2, wherein the solution obtained is filtered and dialyzed, preferably before rotary evaporation, against a 0.22 μm nylon membrane, the rotary evaporation temperature being 50 to 60 ℃.
9. Use of the inhibitor carbon dot for inhibiting amyloid β protein aggregation according to claim 1 for the preparation of a medicament for the treatment of alzheimer's disease.
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