Evaluating The Properties Of A New Penetrating Peptide And Its Application In Intracellular Delivery For Anti-Tumor Drugs

Cell-penetrating peptide(CPP)has been widely studied and applied as trans-membrane carriers for macromolecule drugs in vivo.Proteins that have heparin-binding domain(HBD)could bind heparin/heparin sulfate(HS)and then some of them could be internalized into cells.It was reported that human heparin-binding epidermal growth factor-like growth factor(HB-EGF)and IGF-binding proteins have been successfully developed as CPPs.Midkine(MK)is also a growth factor with an HBD that is involved in a variety of physiological processes,such as development,reproduction,repairment of cells,etc.The HBD derived from the C-terminal domain of human-MK is rich in basic amino acids(TKPCTPKTKAKAKAKKGKGKD,named HMD)which is similar with classical CPPs.In this paper,HMD was fused with the enhanced green fluorescent protein(EGFP)firstly,and then the trans-membrane activity of the fusion protein was studied.First of all,we constructed the recombinant vector EGFP-HMD-pET28a,and then the fusion protein EGFP-HMD was expressed and purified.The cell uptake experiments by fluorescence microscope showed that HMD could transport EGFP into HeLa cells effectively,and its trans-membrane activity is higher than the classical CPP-Tat about 4 times which suggested that HMD is a novel CPP with highly trans-membrane activity.In order to elucidate the trans-membrane mechanism of HMD-carrying macromolecule proteins,the novel trans-membrane peptide HMD was studied in terms of trans-membrane dynamics analysis(the relationship between the trans-membrane efficiency and time、concentration),the transduction efficiency of HMD into various kinds of cells and trans-membrane pathways.The studies showed that:1.HMD penetrates into HeLa cells in a time-and concentration-dependent manner;2.HMD could carry EGFP into a variety of tumor cells and could hardly penetrate membranes into normal cells,showing the trans-membrane variability between tumor cells and normal cells;3.the trans-membrane mechanism of HMD by endocytic pathways inhibitors have shown that HMD is dependent on a variety of endocytosis pathways including caveolin,lipids,macropinocytosis and interacts with cell surface heparin sulfate proteoglycans when carrying EGFP into the cell.Analysis of HMD trans-membrane sequences by multiple mutations in HMD revealed that basic amino acids in HMD play an important role in trans-membrane formation;and it is speculated that theα-helical structure in HMD plays a structural supporting role for binding sites at both ends;the binding sites at both ends also play significant role in penetrating membranes.We further examined the capability of HMD to carry anti-tumor drugs(MAP30,α-MMC,TCS)into cells,and evaluated the anti-tumor activity of fusion proteins.The MTT cytotoxocity assay showed that the introduction of HMD increased the toxicity of anti-tumor drugs on tumor cells,such as the cytotoxicity of MAP30-HMD in HeLa,MGC803 increased by 6.07,5.42 times when compared to MAP30 alone;the cytotoxicity of α-MMC-HMD in HeLa and MGC803 was increased by 9.52 and 7.04 folds respectively when compared withα-MMC alone;compared with TCS alone,the cytotoxicity of TCS-HMD in HeLa and MGC803 was increased by 46.12 and 9.81 times respectively;Inhibition of MRC-5 by anti-tumor drugs fused with HMD is approximately the same as the anti-tumor drugs alone remaining the low toxicity to normal cells.This article has successfully obtained a novel cell-penetrating peptide with highly efficiency-HMD,which has a significant role in the application of multiple anti-tumor drugs into intracellular to exert their efficiency,thus expanding the application of HMD in anti-tumor applications.The study on the molecular mechanism of HMD penetrating into cells and the application in anti-tumor drug transporting will promote the application of drug transporting vector in tumor therapy.