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Cell-penetrating peptide and particles thereof coating hydrophobic molecules and preparation method and application of particles

A membrane-penetrating peptide and hydrophobic technology, applied in chemical instruments and methods, medical preparations of non-active ingredients, peptides, etc., can solve the problems that cannot meet the requirements of imaging or drug loading applications, limited number and types of molecules, and bonding process Complicated and other issues, to achieve the effect of reducing photobleaching, uniform size, and improving signal-to-noise ratio

Inactive Publication Date: 2019-03-26
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The invention solves the technical problems in the prior art that the chemical bonding process between membrane-penetrating peptides and hydrophobic molecules is complex, and the number and types of bonded molecules are limited, which cannot meet the requirements of imaging or drug-loading applications

Method used

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  • Cell-penetrating peptide and particles thereof coating hydrophobic molecules and preparation method and application of particles
  • Cell-penetrating peptide and particles thereof coating hydrophobic molecules and preparation method and application of particles
  • Cell-penetrating peptide and particles thereof coating hydrophobic molecules and preparation method and application of particles

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Synthesis of polypeptide sequences: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: The peptides of the sequence shown in 11 are all composed of common amino acids and prepared by the classic solid-phase synthesis method. After these peptide sequences are purified by liquid chromatography, the purity can reach more than 95%.

[0048] Wherein, the HPLC spectrogram of the purified SEQ ID NO: 4 is shown in figure 1 , the mass spectrum of the sequence: [M+3H] 3+ =831.79.

[0049] The mass spectra of the remaining peptides are as follows:

[0050] The mass spectrum of the sequence SEQ ID NO: 5 of the amphiphilic cell-penetrating peptide: [M+3H] 3+ =706.81

[0051] The mass spectrum of the sequence SEQ ID NO: 6 of the amphiphilic cell-penetrating peptide: [M+6H] 6+ =465.43

[0052] The mass spectrum of the sequence SEQ ID NO: 7 of the amphiphilic cell-penetrating peptide: [M+5H] 5+ =598.18

[0053] The m...

Embodiment 2

[0058] Preparation method of amphiphilic cell-penetrating peptide and nanoparticles coated with hydrophobic molecules

[0059] Take 25 μL of 1 mg / mL aqueous solution of sequence SEQ ID NO: 4, and slowly add it dropwise to 100 μL of 0.25 mg / mL hydrophobic fluorescent molecule Nile Red tetrahydrofuran solution. Good, uniformly sized nanoparticles. Its transmission electron microscope picture is shown in figure 2 (a), DLS diagram see image 3 (a).

[0060] Take 50 μL of 1 mg / mL aqueous solution of sequence SEQ ID NO: 5, and slowly add it dropwise to 100 μL of 0.25 mg / mL hydrophobic delayed luminescence molecule 4CzIPN (Ishimatsu Ryoichi, et al.Solvent effect on thermally activated delayed fluorescence by1,2,3,5 -tetrakis(carbazol-9-yl)-4,6-dicyanobenzene.Journal of Physical Chemistry A(2013)117(27):5607-5612) tetrahydrofuran solution, after it is ultrasonically uniform, add 900μL deionized water to obtain Nanoparticles with good dispersion and uniform size. Its transmission...

Embodiment 3

[0068] Cytotoxicity assay of amphiphilic cell-penetrating peptides and Nile red nanoparticles coated with MTT

[0069] The same number (1.5 × 10 4 / well) cells in a 96-well plate in complete medium, 37°C, 5% CO 2 The cells were cultured for 12 hours under the environment of the environment, and the cells adhered to the wall. Add 10 μL of the above-prepared Nile Red nanoparticles, mix well and then incubate at 37°C, 5% CO 2 Incubate for 24 hours under ambient conditions. After incubation, MTT (5 mg / mL) was added, followed by incubation for 4 h, the upper layer liquid was sucked off, and 100 μL DMSO solution was added. The absorbance of each well was detected at 570 nm by a microplate reader. Model of microplate reader: Tecan Infinite F50.

[0070] The cytotoxicity of amphiphilic cell-penetrating peptide and its coated Nile red nanoparticles was detected by MTT assay. Figure 4 (a) shows that after 24 hours of incubation with the amphiphilic cell-penetrating peptide, the c...

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Abstract

The invention discloses a cell-penetrating peptide and particles thereof coating hydrophobic molecules and a preparation method and an application of the particles and belongs to the technical field of nano biomaterials. The amino acid sequence of the cell-penetrating peptide is X-Y-(r-R)3-(R)-2, wherein X is a peptide chain with at least three amino acid residues and at most 20 amino acid residues; the amino acids in X are separately selected from phenylalanine, leucine, isoleucine, alanine, tryptophan, proline, methionine or histidine; Y is a peptide chain with at most 20 any amino acid residues; r is D arginine; R is L arginine. The amphipathic cell-penetrating peptide and hydrophobic molecules are co-assembled to be self-assembled to form nanoparticles through the hydrophobic interaction of the noncovalent molecules. The nanoparticles can enable the hydrophobic molecules to penetrate cytomembranes quickly with low cell toxicity into the cells. The nanoparticles have a good application prospect in the fields of cytomembrane fluorescence imaging, time resolution imaging, hydrophobic drug coating and the like.

Description

technical field [0001] The invention belongs to the technical field of nano-biological materials, and more specifically relates to an amphiphilic cell-penetrating peptide, nanoparticles coated with hydrophobic molecules, a preparation method and application of the particles. Background technique [0002] The cell membrane is a biological barrier that can prevent fluorescent probe molecules, biomacromolecules, and drug molecules from entering cells. Fluorescent probes that are less permeable to the cell membrane require a longer incubation time to complete the transmembrane process, resulting in a delay in real-time fluorescence detection. Similarly, many biomacromolecules and drug molecules with therapeutic efficacy are also difficult to enter diseased cells to exert biological effects, which greatly reduces the efficacy of drugs and increases the amount of drugs used and potential safety hazards. Therefore, how to make fluorescent probe molecules, biological macromolecules...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K47/69A61K47/64
CPCA61K47/64A61K47/6929C07K7/06C07K7/08C07K2319/03
Inventor 朱锦涛田迪朱泽策
Owner HUAZHONG UNIV OF SCI & TECH
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