New cartilaginous affinity polypeptide sequence, screening method and application thereof

A sequence and cartilage technology, applied in the application field of targeted PEI nanocarriers, can solve the problems of low gene transfection efficiency and the inability of polypeptide sequences to take into account tissue specificity and cell affinity, so as to improve tissue specificity. Effect

Active Publication Date: 2010-11-24
PEKING UNIV THIRD HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology is rarely used in gene therapy of articular cartilage. The first to carry out this research was ROTHENFLUH[26]. The targeting of gene therapy, however, the polypeptide fragments screened mainly target the type II collagen in the matrix around the cartilage, resulting in the modified nano-carriers having too high affinity for the matrix around the cartilage, and unable to extract from the matrix of the cartilage. Detach and enter into chondrocytes, making the vector-mediated gene transfection efficiency too low
It can be seen that the previous methods either only screened at the tissue level or only screened at the cell level, resulting in the acquisition of polypeptide sequences that could not take into account the two requirements of tissue specificity and cell affinity

Method used

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  • New cartilaginous affinity polypeptide sequence, screening method and application thereof
  • New cartilaginous affinity polypeptide sequence, screening method and application thereof
  • New cartilaginous affinity polypeptide sequence, screening method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1 Screening of cartilage-affinity polypeptide sequences by phage display technology

[0102] 1. The first round of screening

[0103] (1) Rabbit synovium and cartilage sample preparation:

[0104] Select 4-week-old healthy rabbits, male or female, and overdose with 100 mg / Kg pentobarbital sodium to anesthetize them to death. The double knee joints of rabbits were separated under aseptic conditions, and the muscles and ligaments around the joints were removed. Under a microscope, the synovial tissue on the surface of the articular cartilage was scraped off with a sharp blade, and the articular cartilage and synovial tissue were cut. PBS (Na 2 HPO 4 10mM, KH 2 PO 4 2mM, NaCl 137mM, KCl 2.7mM, pH 7.4, DecentBio) fully washed 3 times.

[0105] Positive screening dish: Cut rabbit articular cartilage into cartilage slices with a diameter of about 5-10mm and a thickness of 2-3mm, about 20-30 slices, and place them in a 60mm culture dish;

[0106] Negative scre...

Embodiment 2

[0149] Example 2 Modification of polyethylenimine cationic polymers with articular cartilage tissue-specific and chondrocyte-high affinity polypeptides

[0150] 1. Polypeptide synthesis: Synthesize and screen the obtained cartilage-friendly polypeptide, and connect a cysteine ​​(C) at its 3'C end to obtain the corresponding cartilage-friendly polypeptide DWRVIIPPRPSAC (synthesized by Scilight-peptide Inc, China), using FITC (fluorescein isothiocyanate, Fanbo, China) fluorescent dye was used to label the polypeptide. A cysteine ​​residue (C) is linked at the 3' end of the polypeptide in order to covalently modify PEI (25KDa) so that it can be combined with the imino group (-NH 2 ) covalently bound.

[0151] 2. Activation of PEI and coupling of peptides:

[0152] (1) Under a dry nitrogen atmosphere, dissolve 5 mg PEI in 500 μl DMSO (dimethylsulfoxide, Dimethylsulfoxide, sigma) to form a 10 mg / ml solution;

[0153] (2) Add 1 mg of MBS (Maleiimido-benzoic acid Nhydroxysuccinimide...

Embodiment 3

[0157] Example 3 Identification of cartilage-affinity polypeptides for the affinity of rabbit-derived chondrocytes

[0158] In this example, cartilage-affinity phages containing DWRVIIPPRPSA polypeptide fragments were used to detect their affinity to rabbit-derived chondrocytes.

[0159] 1. Rabbit-derived chondrocyte culture:

[0160] (1) Cut out the full-thickness cartilage slices of the rabbit knee joint under aseptic conditions, wash them three times with PBS containing 0.1% (W / V) ampicillin, and cut off non-cartilage tissues such as synovial tissue or fibrous connective tissue;

[0161] (2) Transfer the cleaned cartilage into a 60mm petri dish, add 6ml of 0.2% trypsin (Gibco, USA), digest it in a 37°C incubator for 60min (preferably in a state of stirring), and remove the synovium on the surface of the cartilage and fibroblasts, discard the trypsin, wash 3 times with PBS (pH 7.4) containing 0.1% (W / V) ampicillin;

[0162] (3) Resuspend the cartilage slices with 1ml PBS, ...

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Abstract

The invention discloses a new cartilaginous affinity polypeptide sequence and a screening method thereof. In the invention, according to an improved phage display technique, a cell and tissue layers combined extracorporeal screening method is applied to cartilaginous affinity polypeptide screening for the first time so as to screen out the polypeptide sequence DWRVIIPPRPSA with chondrocyte affinity and tissue specificity, and thus, the drawback of simple cell screening or tissue screening in the prior art is overcome, and the cellular affinity and the tissue specificity are well combined. The invention also relates to the application of the polypeptide in the preparation of a targeted PEI nano-carrier for the gene therapy of mediated articular cartilage diseases, which realize the decoration on the PEI carrier by the cartilaginous affinity polypeptide, offers the cartilaginous tissue specificity and the cellular affinity to the carrier, and realizes the targeted therapy to the cartilage. In addition, the invention takes the chondrocyte itself as the screening target, which has better targeted effect and clinical practical value compared with the mode of taking the matrixes around the chondrocyte as the screening target in the prior art.

Description

technical field [0001] The present invention relates to the preparation and application of biochemical polypeptides, in particular to a new cartilage-affinity polypeptide sequence and its screening method, and the present invention also relates to the use of the polypeptide in the preparation of targeted PEI nanocarriers that mediate gene therapy for articular cartilage diseases The application in belongs to the field of biomedicine. Background technique [0002] Osteoarthritis is a chronic bone and joint disease caused by the degeneration of articular cartilage. It often makes the affected joints swollen, painful, stiff, and limited in movement, eventually leading to limb disability. In fact, osteoarthritis has become one of the leading causes of loss of mobility in older adults. As our country enters an aging society, osteoarthritis will become an increasingly serious problem, causing a huge burden on individual families and the social medical and health system [1, 2]. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/04A61K48/00A61K47/42A61P19/02A61P19/08
Inventor 皮彦斌敖英芳张辛石俊俊朱敬先
Owner PEKING UNIV THIRD HOSPITAL
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