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Cell membrane penetrating conjugates

A technology of conjugates and cells, applied in the direction of hybrid peptides, peptides, chemical instruments and methods, etc.

Pending Publication Date: 2020-07-14
싸이카온코솔루션즈리미티드
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] For the most common genetic diseases, such as cystic fibrosis or muscular dystrophy, effective gene therapy may remain a challenge due to difficulties in delivering genetic material into cells
There is no easy way to deliver genes to the large number of cells in tissues such as lung epithelium or skeletal muscle (Collins et al., Proc. R. Soc. B. Vol. 282. No. 1821. The Royal Society, 2015)

Method used

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Experimental program
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Embodiment 1

[0320] Plasmid / Protein Research -The plasmid containing the AlbG gene shown in SEQ ID NO:19 was obtained by a previously published method (Vetting et al.Acta Crystallogr.Sect.FStruct.Biol.Cryst.Commun.2011:67(3):296- 302, the contents of which are incorporated herein by reference, and specific details are provided below).

[0321] The open reading frame of AlbG was amplified by standard PCR techniques using X albilineans (ATCC 29184; Pieretti et al., BMC Genomics, 2009, 10:616, 1-15) chromosomal DNA as a template. Oligonucleotides AlbGF (5'-ATCCCGCTCATATGCCGGCCAAGACCCTTG-3') and AlbGR (5'-ATCCCGCTCTCGAGTCAATCGGACAGCTCGATATC-3') containing NdeI and XhoI restriction sites, respectively, were used. The PCR fragment was cloned into pET-28a(+) and recombinant AlbG with a thrombin-cleavable N-terminal His6 tag was expressed in E. coli strain BL21(DE3). For shake flask growth, 10 ml of overnight culture was inoculated into 1 liter of Luria Broth supplemented with kanamycin (35 μg / ...

Embodiment 2

[0327] Toxicity assay - An industry standard MTT assay (from Sigma Aldrich) was performed to analyze the toxicity of β-helical protein-cytotoxic drug conjugates to mammalian cells such as HeLa cells. Cells were cultured by using standard protocols. One million cells were seeded in a confocal plate (1 cm dish) and grown for 6-8 h. A mixture of cytotoxic drug and EfsQNR protein (mixed in a 2:1 ratio) was added to the cells and incubated at room temperature for 15 min to 24 h to 72 h. After incubation, cells were washed with PBS and treated with MTT, and further incubated for 24 hours. Cells were then washed and analyzed for absorbance at 570 nm. Viable cells with active metabolism convert MTT to the purple formazan product with an absorbance maximum near 570nm. Dead cells cannot convert MTT to formazan, therefore, by analyzing the absorbance value at 570nm, the percentage of live cells for a given protein or any other molecule can be calculated.

Embodiment 3

[0329] Labeling proteins with fluorescent dyes- Fluorescent dyes are considered as an example of functional molecules to study the cell membrane penetration ability of conjugates. Recombinant proteins are labeled with dyes by performing a series of reactions under dark conditions. The protein to be labeled is collected in PBS buffer (1X and pH 7.3), and the concentration of the dye collected is 2-3 times that of the protein. Proteins were added to 0.1 M sodium carbonate buffer (pH 8.5) followed by dye addition. The dye was added very slowly (3 μl each) to the buffer containing the protein, which was kept on ice with occasional shaking. The resulting solution was wrapped in aluminum foil to keep out any light. The solution was stirred at room temperature for 1 hour and then purified by gel filtration by passing it through a desalting column or a PD 10 column with 1X PBS buffer. Characterize the resulting labeled protein using UV-Vis spectrophotometry to determine the dye t...

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Abstract

A cell penetrating conjugate comprising a recombinant beta helical protein linked to a functional molecule wherein the beta helical protein length is in the range of from 5nmto 25nm, suitably, from 10nm to 15nm and width is in the range of from 1nmto 5nm, suitably,from 1nm to 3nm.Processes for preparing said conjugates and uses thereof are also disclosed.

Description

technical field [0001] The present disclosure broadly relates to the field of delivery of drugs to the cytoplasm, and in particular discloses conjugates comprising recombinant proteins linked to functional molecules for penetrating cell membranes, methods for preparing the conjugates and uses thereof. Background technique [0002] The cell membrane is a semipermeable membrane that separates the internal environment of a cell from its external environment. The membranes of prokaryotic and eukaryotic cells, although differing in some properties and compositions, all contain a semipermeable bilayer structure of phospholipids. The semipermeability of the cell membrane makes it selective for the types of molecules that can penetrate it. Those molecules capable of penetrating cell membranes hold promise for cell labeling, cell penetration, cell delivery, drug uptake, gene therapy, and many other applications involving cell membrane penetration. [0003] There are certain peptide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00
CPCC07K19/00A61K47/65A61K47/60A61K47/62
Inventor N·杰姆桑加米塔
Owner 싸이카온코솔루션즈리미티드
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