Research On Properties Of Cell-penetrating Peptides Targeting Tumor Cells

Targeted cancer therapy is currently a hot research for anticancer drugs.Due to the natural barrier of cell membrane,some antitumor protein drugs cannot enter cells and develop its pharmacological effects.Cell-penetrating peptides is cause for concern,since it has the ability to efficiently transport of exogenous molecules inside cell.Both tumor cells and normal cells,CPP does not have the ability to selectively kill,therefore CPPs maybe have a high cytotoxic in the treatment of cancer.Then looking for a kind of efficient tumor targeting drug delivery carrier is very important.Heparin binding domain(HBD)is from the c terminal of the human superoxide dismutase(SOD),as a human-derived peptides have a efficient ability of penetrating the membrane carrying exogenous protein drug.However,it lack of tumor targeting transport.As a common target of anti-tumer drugs,Epidermal growth factor receptor(EGFR)is overexpressed in a variety of tumor cells.Vaccinia Growth Factor(SX)and Epidermal Growth Factor(EGF)has a similar spatial structure,and having a specific ability of binding EGFR.SX3 from the third loop sequence of SX is a major recognition region of EGFR.Through the integration between SX3 and HBD,obtain a highly efficient drug delivery carrier which is targeting tumor cells.Enhanced green fluorescent protein as a marker,building the expression vectors of pET28a-EGFP-SX3-HBD by molecular biological techniques.pET28a-EGFP-SX3-HBD converted to BL21(DE3)for expression.Purified EGFP-SX3-HBD fusion protein(ES3H)by Ni2+ affinity chromatography.Confocal laser scanning microscope and flow cytometry analysis showed that,ES3H has obvious penetrating activity against HeLa,Bcap,Caski,A549 compared with the EGFP-HBD(EH)of lacking SX3.Further research of ES3H penetrating properties shows that,similar to the EH,penetrating activity of ES3H has time and concentration-dependent manner.EGFR masking data showes that EGFR play an important role in the target recognition of ES3H.Studies on inhibitors showed ES3H achieves transmembrane transduction by the way of macropinocytosis.By means of genetic engineering,successfully constructed SX3-Tat expression vector.Compared with Tat,the cell-penetrating efficiency of SX3-Tat against tumor cells has been significantly improved.The above data shows,SX3 is a universal tumor cell targeting sequences.Its combination of SX3 and HBD gives the HBD fusion protein ability to target tumor cells,and then build VP3-SX3-HBD expression vector to express and purification,obtained purified protein drugs VP3-SX3-HBD.MTT assay showed,the drug against HeLa has obvious effect of tumor destruction,at the same time toxicity to 293 cells decreased.