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D-configuration polypeptide with brain tumor targeting and tumor tissue penetration ability and its gene delivery system

A brain tumor and configuration technology, applied in the field of pharmacy, to achieve the effect of prolonging the survival period and improving the efficiency of gene transfection

Active Publication Date: 2017-02-15
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently, there is no information about the D (RPPREGR) sequence polypeptide modified non-viral gene delivery system and its transfection efficiency evaluation in vivo and in vitro and the report of its anti-glioma effect

Method used

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  • D-configuration polypeptide with brain tumor targeting and tumor tissue penetration ability and its gene delivery system
  • D-configuration polypeptide with brain tumor targeting and tumor tissue penetration ability and its gene delivery system
  • D-configuration polypeptide with brain tumor targeting and tumor tissue penetration ability and its gene delivery system

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Experimental program
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Effect test

Embodiment 1

[0032] Preparation and characterization of RPPREGR

[0033]Using the solid-phase synthesis method, deprotect the PAM-Boc resin with trifluoroacetic acid (TFA) for 1 minute, twice, and use Boc to protect the D-configuration amino acid to react in turn. The resin was washed with DMF, DCM / MeOH (1 / 1) and dried in vacuo. Put the resin into a polypeptide cutting tube, add an appropriate amount of P-cresol, then pass through HF, stir in an ice bath for 1 hour, remove the HF in the tube under reduced pressure after the reaction, wash the precipitate with ice ether for 3 times, and wash the residual precipitate with 20% acetonitrile After dissolving, spin evaporate, use acetonitrile / water (containing 0.1% TFA) system to separate and purify, HPLC and ESI-MS characterize the purity and molecular weight of RPPREGR, its HPLC spectrum and mass spectrum are as follows figure 1 As shown, the molecular weight of RPPREGR is 866.9, which is consistent with the calculated results.

Embodiment 2

[0035] Preparation of RPPREGR-PEG-PEI

[0036] Dissolve 20mg NHS-PEG-Mal and 11.3mg RPPREGR polypeptide (molar ratio 1:1.3) in 1mL DMF, slowly drop into 300μl DMF, stir at room temperature for 1h, HPLC detects the formation of RPPREGR-PEG-Mal, the above reaction The solution was diluted one-fold with pure water, then transferred to Sephadex G-15 gel column, separated and purified by AKTA Explorer1100 Series [mobile phase pure water; flow rate 1ml / min], and the RPPREGR-PEG-Mal fraction was collected and freeze-dried.

[0037] Dissolve 15mg of PEI in 1.5mL of 0.2M PBS (pH=7.4) buffer solution, adjust the pH to about 7.0 with HCL, and dissolve 8.25mg of RPPREGR-PEG-Mal (molar ratio 1:5) in 0.5mL of 0.2 M PBS (pH=7.4) buffer solution, stirred and added to the above PEI reaction solution, stirred overnight at room temperature, ultrafiltered (Mw=10kDa, AmiconUtro-4mL, Millipore) 5 times, and freeze-dried to obtain RPPREGR-PEG- PEI.

Embodiment 3

[0039] Preparation and Characterization of RPPREGR-PEG-PEI / pDNA

[0040] pGL 4.2 Plasmid solution and carrier material solution were mixed in equal volumes, the final concentration of pDNA was 40 μg / mL, vortexed immediately for 30 s, and left at room temperature for 30 min to obtain a freshly prepared complex solution. The concentration of carrier material depends on N / P, and its calculation formula is N / P=7.53×PEI (g) / DNA (g), the particle size and Zeta potential of each sample were measured with the NanoZS particle size analyzer of Malvern, the results are as follows figure 2 As shown, at NP=12, PEG-PEI / pGL 4.2 and RPPREGR-PEG-PEI / pGL 4.2 The particle size is about 200nm, and the potential is positive.

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Abstract

The invention belongs to the field of pharmacy and relates to a D-configuration polypeptide and a gene delivery system of the D-configuration polypeptide and particularly relates to the D-configuration polypeptide which has high combining activity with neuropilin NRP-1 and has the brain tumor targeting and tumor tissue penetrating capabilities. The D-configuration polypeptide provided by the invention can mediate a nano delivery system to deliver drugs to tumors in a targeted manner for realizing targeted treatment of brain in-situ glioma. In vivo and in vitro experiments show that the genetic vector modified by the D-configuration polypeptide can remarkably improve the gene transfection efficiency. The genetic vector entrapping the therapeutic gene pORF-hTRAIL can remarkably prolong the lifetime of a nude mouse of brain glioma in-situ mode.

Description

technical field [0001] The invention belongs to the field of pharmacy, and relates to a D-configuration polypeptide and its gene delivery system, in particular to a D-configuration polypeptide with high binding activity to neuropilin NRP-1, brain tumor targeting and tumor tissue penetrating ability and its modification gene delivery system. Background technique [0002] According to reports, glioma is one of the most common brain tumors; due to its high degree of malignancy, easy recurrence after surgery, and rapid invasion, the median survival time of patients is less than 16 months. For the successful treatment of brain tumors, drugs or therapeutic genes must not only cross the blood-brain barrier (BBB) ​​but also overcome the blood-brain tumor barrier (BBTB) to reach the tumor site. Studies have shown that specific receptors expressed on tumor cells are effective targets for tumor-targeted drug delivery, and tumor-targeted therapy mediated by them is an effective means o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C12N15/87A61K48/00A61K47/42A61P35/00
Inventor 刘敏王晶陆伟跃谢操雷杨谢作旭
Owner FUDAN UNIV
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