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CN-121978070-A - Nanoparticle with AIE characteristics and application thereof in LSD1 inhibitor detection

CN121978070ACN 121978070 ACN121978070 ACN 121978070ACN-121978070-A

Abstract

The invention discloses a nanoparticle with AIE characteristics and application thereof in LSD1 inhibitor detection, belonging to the field of LSD1 inhibitor screening; the preparation method of the nanoparticle comprises the steps of dissolving TPE powder in chromatographic pure CH 2 Cl 2 to obtain an oil phase solution, dissolving recombinant H3K9me2 polypeptide in ultrapure water to obtain an aqueous phase solution, mixing and stirring the aqueous phase solution and the oil phase solution to form a primary emulsion, performing ultrasonic crushing treatment on the primary emulsion to obtain a nanoemulsion, stirring and blowing nitrogen synchronously with the nanoemulsion to remove an organic solvent to obtain AIENP suspension, filtering and centrifugally purifying the AIENP suspension to obtain the nanoparticle with AIE characteristics. The feasibility of the system for LSD1 inhibitor screening was verified by pre-incubating LSD1 with a gradient concentration of LSD1 inhibitor followed by addition of AIENP system, detection of fluorescence intensity and calculation of IC50 value for LSD1 inhibitor.

Inventors

  • HAN CHAO
  • MENG LIN
  • LUO JIANGUANG

Assignees

  • 中国药科大学

Dates

Publication Date
20260505
Application Date
20260206

Claims (10)

  1. 1.A method for preparing nanoparticles having AIE properties, comprising the steps of: Dissolving TPE powder in chromatographic pure CH 2 Cl 2 to obtain oil phase solution, dissolving recombinant H3K9me2 polypeptide in ultrapure water to obtain water phase solution; mixing and stirring the aqueous phase solution and the oil phase solution to form a primary emulsion, and then performing ultrasonic crushing treatment on the primary emulsion to obtain a nano emulsion; stirring and blowing nitrogen synchronously with the nano emulsion to remove the organic solvent to obtain AIENP suspension; Filtering AIENP suspension, centrifuging and purifying to obtain the nano-particle with AIE characteristic.
  2. 2. The method for preparing the nanoparticle with AIE characteristics according to claim 1, wherein the amino acid sequence of the recombinant H3K9me2 polypeptide is shown in SEQ ID NO. 1.
  3. 3. The method of claim 1, wherein the volume ratio of the aqueous phase solution to the oil phase solution is 10:1.
  4. 4. The method for preparing nanoparticles with AIE properties according to claim 1, wherein the ultrasonic power of the ultrasonic disruption treatment is 100W, the ultrasonic time is 2 s, the interval time is 3 s, and the total ultrasonic time is 10-15 min.
  5. 5. The method of claim 1, wherein the filtration is performed using a PVDF membrane of 0.22 μm.
  6. 6. A nanoparticle having AIE properties, prepared by the method of any one of claims 1-5.
  7. 7. Use of the nanoparticle with AIE properties of claim 6 for screening LSD1 inhibitors.
  8. 8. Use of the nanoparticle with AIE properties of claim 6 for detecting LSD1 concentration.
  9. 9. A method of screening for LSD1 inhibitors comprising: resuspending the nanoparticle of claim 6 having AIE properties to provide AIENP stock solution; LSD1 solutions were incubated separately mixed with gradient concentrations of LSD1 inhibitor, followed by the addition of AIENP stock solutions, detection of fluorescence intensity changes, and calculation of IC 50 values for screening LSD1 inhibitors.
  10. 10. A method for quantitatively detecting LSD1 concentration, comprising: resuspending the nanoparticle of claim 6 having AIE properties to provide AIENP stock solution; mixing AIENP stock solution with LSD1 solution to be tested, measuring fluorescence intensity, and obtaining the concentration of LSD1 according to the linear relation between the fluorescence intensity and the concentration of LSD 1.

Description

Nanoparticle with AIE characteristics and application thereof in LSD1 inhibitor detection Technical Field The invention belongs to the field of LSD1 inhibitor screening, and particularly relates to a nanoparticle with AIE characteristics and application thereof in LSD1 inhibitor detection. Background Aggregation-induced emission (AIE) materials have become a research hotspot in the field of biological detection due to their unique optical properties of weak or no fluorescence in a dispersed state, while fluorescence is significantly enhanced in an aggregated state. Tetraphenyl ethylene (TPE) as a typical AIE molecule has good photostability and biocompatibility, and its aggregation-state fluorescence enhancement effect provides an ideal optical signal carrier for constructing a high-sensitivity biological detection system. Histone lysine specific demethylase 1 (Histone lysine-SPECIFIC DEMETHYLASE 1, LSD 1) is used as the first discovered histone demethylase, and participates in various biological processes such as cell proliferation, differentiation, tumorigenesis and development by regulating the single and double methylation levels of histones H3K4me1/2 and H3K9me1/2, and is an important target for diagnosing and treating malignant tumors. The LSD1 inhibitor is researched and developed, so that the epigenetic regulation and control function of histone demethylation of the LSD1 inhibitor can be accurately blocked, the silencing state of cancer suppressor genes in tumor cells can be effectively reversed, proliferation, invasion and metastasis of the tumor cells can be radically inhibited, the tumor immunosuppression microenvironment can be improved, the drug resistance dilemma of immune checkpoint inhibitors can be relieved, a brand new targeting treatment strategy is provided for refractory tumors which are disabled in traditional treatment and immunotherapy, and the blank of clinical application in the field of epigenetic targeting anticancer is powerfully filled. The existing LSD1 inhibitor screening method mainly comprises two methods of direct detection and by-product detection. The direct detection method is to detect by MS, TRF and other methods. The most common method based on byproduct detection is quantification of byproduct H 2O2 as a method of screening for LSD1 inhibitors by fluorescent probe AR-HRP system. However, the existing screening methods for LSD1 are not perfect. The LSD1 inhibitor can be directly detected by using an MS method, but the defects of low ionization efficiency and poor ionization characteristics of the demethylated histone peptide exist, and the ionization process is easily influenced by various factors. Screening for LSD1 inhibitors based on detection of H 2O2 production by the AR-HRP system is one of the most common but may lead to false positive results because AR can also be converted to Resorufin and HRP by exposure to hydrogen-free ambient light and some compounds exhibit HRP inhibition, reducing H 2O2 production. Disclosure of Invention In view of the shortcomings of the prior art, the present invention aims to provide a nanoparticle with AIE properties and its application in LSD1 inhibitor detection, which solve the problems in the prior art. The aim of the invention can be achieved by the following technical scheme: a method of preparing nanoparticles having AIE properties, comprising the steps of: Dissolving TPE powder in chromatographic pure CH 2Cl2 to obtain oil phase solution, dissolving recombinant H3K9me2 polypeptide in ultrapure water to obtain water phase solution; mixing and stirring the aqueous phase solution and the oil phase solution to form a primary emulsion, and then performing ultrasonic crushing treatment on the primary emulsion to obtain a nano emulsion; stirring and blowing nitrogen synchronously with the nano emulsion to remove the organic solvent to obtain AIENP suspension; Filtering AIENP suspension, centrifuging and purifying to obtain the nano-particle with AIE characteristic. Further, the amino acid sequence of the recombinant H3K9me2 polypeptide is shown as SEQ ID NO. 1. Further, the volume ratio of the aqueous phase solution to the oil phase solution is 10:1. Further, the ultrasonic power of the ultrasonic crushing treatment is 100W, the ultrasonic time is 2 s, the interval time is 3s, and the total ultrasonic duration is 10-15 min. Further, the filtration employed a PVDF filter of 0.22 μm. The nanometer particle with AIE characteristic is prepared by the preparation method. Use of the nanoparticle having AIE properties described above for screening LSD1 inhibitors. Use of the nanoparticle having AIE properties described above for detecting LSD1 concentration. A method of screening for LSD1 inhibitors comprising: resuspending the nanoparticles with AIE properties described above to obtain AIENP stock solution; LSD1 solutions were incubated separately mixed with gradient concentrations of LSD1 inhibitor, followed by the addition of AIENP