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Application and preparation method of erythrocyte bionic nanomaterial of PCM polypeptide combined with KALA polypeptide

A bionic nanometer and red blood cell technology, applied in the field of biomedicine, to achieve the effect of concentrated action, effective heart enrichment function, and increased blood half-life

Pending Publication Date: 2021-11-05
THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This scheme discloses the combination of PCM with targeting function and cell penetrating peptide, but this combination can only use PCM to achieve the targeting effect, and needs to rely on the concentration of the drug to import the drug into the cell

Method used

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  • Application and preparation method of erythrocyte bionic nanomaterial of PCM polypeptide combined with KALA polypeptide
  • Application and preparation method of erythrocyte bionic nanomaterial of PCM polypeptide combined with KALA polypeptide
  • Application and preparation method of erythrocyte bionic nanomaterial of PCM polypeptide combined with KALA polypeptide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The composition is the application of the nanometer material composed of PCM polypeptide and KALA polypeptide combined and inserted into the red blood cell membrane in the treatment of cardiovascular diseases.

[0030] In this application, PCM has a heart-targeting function and can carry KALA polypeptide to the target position of cardiovascular disease. KALA polypeptide maintains a secondary structure in an environment of pH=7.4, and cooperates with PCM to form a stable delivery substance. KALA also has the function of curling The hydrophobic amino acid and the exposed amino acid, the exposed amino acid can mediate the rapid phagocytosis of KALA by cells. Compared with TAT in the prior art, it is independent of concentration and has higher phagocytosis efficiency. After KALA enters the lysosome, as the pH drops, the secondary structure of the KALA polypeptide is destroyed, and the hydrophobic amino acids of KALA are exposed and fused with the lysosome membrane, which med...

Embodiment 2

[0032] PCM / KALA peptide synthesis

[0033] (1) Raw material protected amino acids Fmoc-Glu(otbu)-OH, Fmoc-Cys(trt)-OH, Fmoc-Ala-oh, Fmoc-Lys(boc)-oh, Fmoc-Leu-oh, Fmoc-His(trt )-oh, Fmoc-Trp(boc)-oh, Fmoc-Ser(tbu)-oh, Fmoc-Gly-oh, Fmoc-Pro-oh, Fmoc-Val-oh, Fmoc-Thr(tbu)-oh, Fmoc -Arg(pbf)-oh

[0034] Starting resin: fmoc-Ala-wang Resin

[0035] Condensing agent and organic base: HBTU, NMM

[0036] Solvents: DMF, DCM, methanol, hexahydropyridine.

[0037] (2) Equipment and instruments:

[0038] Glass reaction column, vacuum water pump, dry heater, vulgar large-capacity centrifuge, constant temperature shaker, vacuum drying dish

[0039] (3) Preparation of various reagents:

[0040] Preparation of Kaiser test reagent

[0041] Liquid A: 80% phenol + 20% absolute ethanol Liquid B: redistilled pyridine Liquid C: 5g ninhydrin + 100ML absolute ethanol

[0042] Preparation of deprotection solution

[0043] 20% Hexahydropyridine + 80% DMF

[0044] Preparation of peptide cutti...

Embodiment 3

[0063] Red blood cell membrane (RBCM) preparation

[0064] The erythrocytes are derived from C57BL / 6 mice (B6 mice), and the process is as follows: collect whole blood of B6 mice, centrifuge at 3000-5000 rpm for 15 minutes to remove white blood cells, platelets and serum, and obtain erythrocytes. Add 250 μL of red blood cells to 950 μL ultrapure water, ice bath for 30-60 min, add 20x PBS to adjust the osmotic pressure to lx, mix well, centrifuge at 14000 rpm for 10 min, discard the supernatant, add 950 μL ultrapure water to resuspend, and ice bath again, Adjust the osmotic pressure, centrifuge, and circulate until the supernatant has no hemoglobin, and collect the precipitate ( figure 2 ). The collected precipitate is the red blood cell membrane (Red Blood Cell Membrane, RBCM). The concentration of membrane proteins in RBCM was determined by the BCA method.

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Abstract

The invention discloses an application of an erythrocyte bionic nanomaterial of PCM polypeptide combined with KALA polypeptide in treatment of cardiovascular diseases, and also discloses a preparation method of the erythrocyte bionic nanomaterial. The method comprises the following steps: obtaining a proper quantity of PCM, performing combining with KALA, and performing fusing into a fusion peptide; enabling the fusion peptide to be subjected to amino acid protection operation treatment, so as to obtain PCM / KALA polypeptide; enabling the PCM / KALA polypeptide to be combined on the surface of an erythrocyte membrane (RBCM); and enabling the PCM / KALA polypeptide combined with the erythrocyte membrane to pass through a polycarbonate membrane with the pore diameter of 400nm by utilizing an Extrusion method, so as to construct the RBCM-PCM / KALA erythrocyte bionic nanoparticles. The erythrocyte bionic nanomaterial prepared by the method can be efficiently and rapidly introduced into cells without depending on concentration, and compared with the prior art, the erythrocyte bionic nanomaterial also has a heart enrichment function.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the application and preparation method of red blood cell bionic nanomaterials composed of PCM polypeptide and KALA polypeptide. Background technique [0002] Cardiovascular disease is the number one killer of human diseases. In recent years, the development of nanotechnology has provided new opportunities for the diagnosis and treatment of cardiovascular-related diseases. Through nanotechnology, drugs can be released slowly and sustainably in the heart, achieving the purpose of long-term treatment. Therefore, how to effectively deliver nanomedicine to cardiomyocytes is the key. [0003] PCM polypeptide (sequence WLSEAGPVVTVRALRGTGSW) has a certain cardiac targeting function. However, the targeting efficiency of PCM is limited by two points: 1) PCM cannot mediate the rapid phagocytosis of nanomaterials by cardiomyocytes, and only provides affinity with cardiomyocytes, so it...

Claims

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Application Information

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IPC IPC(8): A61K47/64A61K47/51A61K38/16A61P9/00B82Y5/00B82Y40/00
CPCA61K47/64A61K47/51A61K38/16A61P9/00B82Y5/00B82Y40/00
Inventor 陈一杰陈梦纯姜琦诸海燕诸葛德力王乐丹颜林志陈钢褚茂平文斌
Owner THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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