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Gene delivery system of targeting brain capillary endothelial cell

A technology of capillaries and endothelial cells, applied in the field of pharmaceutical preparations, to avoid safety problems, improve modifiability, and improve structural stability

Inactive Publication Date: 2014-01-15
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] At present, there are no reports on the gene delivery system using TB short peptide to modify PAE-C32 targeting brain capillary endothelial cells

Method used

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  • Gene delivery system of targeting brain capillary endothelial cell
  • Gene delivery system of targeting brain capillary endothelial cell
  • Gene delivery system of targeting brain capillary endothelial cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, TB peptide and brain capillary endothelial cell binding test

[0057] Synthesize short peptides TB1, TB2, and TB3 labeled with 5-carboxyfluorescein (5-FAM, green fluorescence), and each short peptide molecule is labeled with one 5-FAM. BCEC cells were seeded in a 24-well plate at a density of 10,000 cells per well and cultured for 2 days. The confluence was observed under a microscope to be about 80% for cell binding experiments. Remove the culture medium, add 300 μL TB short peptide solution (containing 10% fetal bovine serum) with a concentration of 48 nmol / mL, incubate at 37°C for 6 hours, wash with ice-cold PBS for 3 times, and measure the short peptides TB1, The combination of TB2, TB3 and BCEC. The result is as figure 1 It was shown that TB1, TB2, and TB3 short peptides can all bind to BCEC very well, and there is no significant difference in the binding positive rate and fluorescence intensity, indicating that TB2 peptide and TB3 peptide have simi...

Embodiment 2

[0058] Embodiment 2, TB1, the cell binding saturation experiment of TB2 peptide

[0059] Using Na 125 Iiodinated TB1 and TB2 peptides were separated by G25 gel chromatographic column to remove free 125 I. BCEC cells were seeded in a 24-well plate at a density of 10,000 cells per well and cultured for 2 days. The confluence of about 80% was observed under a microscope for the binding saturation test. Remove the culture medium, add 300μL containing different concentration 125 I-TB in PBS solution (pH 7.4), make 125 The final concentrations of I-TB were 0.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100, 500, and 1000 nM, cultured at 4°C for 120 min, washed three times with ice-cold PBS, lysed with 200 μL 1N NaOH per well, and then dissolved in 100 μL 2N HCL. and, determine 125 I radioactive intensity, and the BCA method was used to measure the protein content of cells, and each experiment was repeated 4 times. Experimental results Graph Pad Prism 5.0 was used for binding saturation analy...

Embodiment 3

[0060] Embodiment 3, TB1, the pharmacokinetics and tissue distribution of TB2 peptide

[0061] Pharmacokinetics: using Na 125 Iiodinated TB1, TB2 peptide, SD rat intravenous injection containing 80μCi 125 I-TB solution in PBS (0.4mL / rat), blood collection 0.2mL at regular intervals (5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes). Tissue distribution: The administration method is the same as above, the rats are killed at regular intervals (5 minutes, 60 minutes), perfused with Hepes-Ringer buffer solution at 4°C, the brain, cerebellum, heart, liver, spleen, lung, and kidney are quickly weighed, and the brain capillaries are separated. Vascular and brain parenchyma. The gamma counter measures the radioactive intensity in each blood sample and tissue, and calculates the pharmacokinetic parameters and tissue distribution of TB peptide. The amount of TB peptide uptake by each organ is expressed as the percentage of injected dose per gram of tissue (%ID / g t...

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Abstract

The invention belongs to the field of pharmaceutical preparations, and relates to a gene delivery system of a targeting brain capillary endothelial cell mediated by a short peptide. The gene delivery system comprises a targeting functional group, a gene medicament and a carrier, wherein the targeting functional group is a short peptide; the carrier is poly beta-amino ester and polyethylene glycol-poly beta-amino ester; the short peptide is in covalent connection with the polyethylene glycol; and the gene medicament and the carrier form the nano gene delivery system in an entrapment or adsorption manner. By using the gene delivery system, a gene is delivered into a brain capillary endothelial cell in an active targeting manner, so that the brain capillary endothelial cell expresses the required secretion type medicament protein and further secretes and delivers the protein into the brain, thereby realizing treatment of neurodegenerative diseases of the brain.

Description

technical field [0001] The invention belongs to the field of pharmaceutical preparations, and relates to a gene delivery system targeting brain capillary endothelial cells, in particular to a short peptide-mediated gene delivery system targeting brain capillary endothelial cells. Background technique [0002] With the aging of human society, the incidence of neurodegenerative diseases is increasing year by year, and has become one of the major diseases that endanger human life and health. Because the blood-brain barrier (BBB) ​​hinders the delivery of chemical drugs, protein peptides and gene drugs in the brain, it seriously affects the therapeutic effect of brain diseases. At present, there are no effective clinical treatments and drugs for neurodegenerative diseases. . Researchers in this field focus on gene therapy, which may be the most promising and challenging research topic to overcome and cure neurodegenerative diseases. [0003] The study of brain-targeted nano-dr...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K47/42A61K47/34A61K38/18A61P25/28
Inventor 庞志清蒋新国高会乐苏婧晗钱勇魏彦
Owner FUDAN UNIV
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