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Stapled-RGD polypeptide, and applications thereof in tumor targeting delivery

An anti-tumor drug, cyclic peptide technology, applied to stapled-RGD polypeptide and its application in tumor-targeted delivery, can solve problems such as low efficiency and achieve the effect of strong penetrating ability

Inactive Publication Date: 2017-05-24
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The nano drug delivery system has the advantages of high drug loading and long circulation time in the body. It can use the EPR effect of the tumor to passively enrich the drug in the tumor site, but the efficiency is low.

Method used

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  • Stapled-RGD polypeptide, and applications thereof in tumor targeting delivery
  • Stapled-RGD polypeptide, and applications thereof in tumor targeting delivery
  • Stapled-RGD polypeptide, and applications thereof in tumor targeting delivery

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Preparation of sRGD, sRGD-FAM and sRGD-antitumor drug complex, sRGD-PEG-PLA

[0066] 1. Preparation of sRGD

[0067] A circular stapledRGD polypeptide (sRGD) with the amino acid sequence c(XRGDX)GSSGC was prepared by solid-phase synthesis. Wherein, X=R8 / S8 or R5 / S5, two Xs are connected by Grubbs catalyst catalyzed olefin metathesis reaction;

[0068] The Rink-Amide-MBHA resin was deprotected with 20% piperidine N,N-dimethylformamide (DMF) for 15 min twice, and the Fmoc-protected amino acid was dissolved in 0.45M HBTU and HoBt (solvent is DMF), React at room temperature for 45 minutes, wash with DMF, remove Fmoc protection with 20% piperidine, and react sequentially according to the amino acid sequence. For the special amino acid R8 / S5, the amount of amino acid, HBTU and HoBt is halved, and the reaction time is extended to 2h. After the sequence reaction was completed, the olefin metathesis reaction was catalyzed by 10 mM Grubbs first-generation catalyst (1...

Embodiment 2

[0078] Example 2 sRGD and integrin protein binding activity test

[0079] The measuring instrument is a Biacore T100 surface plasmon resonance instrument, the buffer is PBS, and the measuring temperature is constant at 25°C. Integrin α V beta 3 Proteins were immobilized on the chip, and sRGD and cRGD were measured with integrin α by the direct method V beta 3 The binding constant of a protein is determined in two steps:

[0080] (1) Chip preparation: After the surface of the chip is activated by EDC / NHS, add integrin α V beta 3 The protein reacts with it, and the unlinked integrin α is washed away after the reaction V beta 3 protein and detect its connection amount;

[0081] (2) Determination and calculation: PBS buffer solution was used to prepare different concentrations of sRGD polypeptide for sample injection and detection. The instrument measures the binding response value (unit is RU) under different sRGD polypeptide concentrations, and calculates the binding co...

Embodiment 3

[0084] Example 3 In vitro cell targeting verification of sRGD

[0085] 1. In vitro targeting test of sRGD-FAM on glioma cell U87

[0086] Take monolayer cultured glioma cells (U87) in the logarithmic growth phase, digest the monolayer cultured cells with 0.25% trypsin, prepare a single cell suspension with DMEM culture medium containing 10% fetal bovine serum, and dilute each Hole 1×10 5 Cells were inoculated in a 12-well culture plate with a volume of 1 mL per well, and the culture plate was moved into a carbon dioxide incubator at 37°C and 5% CO 2 After culturing for 24 hours under saturated humidity conditions, prepare sRGD-FAM, cRGD-FAM, and FAM solutions at a concentration of 5 μM with DMEM culture solution containing 10% fetal bovine serum; suck out the culture solution in the culture plate, and add the above solutions respectively , Incubate at 37°C for 4h, and discard the supernatant. Wash three times with PBS solution, fix the cells with formaldehyde fixative, stai...

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Abstract

The invention belongs to the field of pharmacology, and relates to stapled-RGD polypeptide, and applications thereof in tumor targeting delivery. Multifunctional targeting polypeptide molecule stapled-RGD with high combination activity with integrin and biological membrane barrier penetration capacity is designed and prepared based on binding peptide cyclization technology; and preparation of fluorescein, drugs, and high molecule carriers modified by the multifunctional targeting polypeptide molecule stapled-RGD, and applications of the fluorescein, the drugs, and the high molecule carriers in tumor imaging and construction of targeted therapy targeted drug delivery systems are disclosed. It is shown by results that specific uptaking of model drugs carried by stapled-RGD by positive cells, tumor mimicry vessels, and tumor ball tissues of expressed integrin is realized, higher tumor targeting capacity, imaging functions, and membrane barrier cell penetration capacity are achieved; nano targeted drug delivery systems constructed by the high molecule carriers modified by stapled-RGD can be used for delivering carried model drugs to target tissues, antitumor drug effect is improved obviously; and stapled-RGD possesses a promising application prospect in mediating drugs, nano targeted drug delivery system membrane barrier penetration, active target searching, tumor diagnosis, and targeted therapy.

Description

technical field [0001] The invention belongs to the field of pharmacy, and relates to a stapled-RGD polypeptide and its application, in particular to a stapled-RGD polypeptide and its application in tumor-targeted delivery, especially to a tumor-targeting neovascularization, tumor-mimicking blood vessel and tumor cells, And it has multifunctional targeting polypeptide molecules across biomembrane barriers, especially the blood-brain barrier (BBB) ​​and blood-brain tumor barrier (BBTB), as well as its modified complexes, nano-drug delivery systems and their application in peripheral tumors and brain tumors. use in diagnosis and treatment. Background technique [0002] According to the data, tumor is a disease that seriously threatens human life and health, and its mortality rate ranks first among all diseases. As the main means of tumor drug therapy, traditional chemotherapy has defects such as poor selectivity to tumor tissue, high toxicity, narrow therapeutic window, and m...

Claims

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Application Information

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IPC IPC(8): C07K7/06A61K47/42A61K49/00A61K9/51A61K9/127A61K9/107A61P35/00
CPCA61K9/107A61K9/1271A61K9/1273A61K9/5146A61K31/337A61K31/4745A61K31/704A61K47/42A61K49/0056C07K7/06
Inventor 陆伟跃陈溪山谢操阮慧瞳
Owner FUDAN UNIV
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