| In the past three decades,a class of peptides called cell-penetrating peptides(CPPs)has been discovered,which have transmembrane activity.Generally,CPPs consist of 5-30 amino acids.They can be divided into cationic penetrating peptides,amphipathic penetrating peptides and hydrophobic penetrating peptides.These peptides can be used as vectors to deliver drugs efficiently.The drugs include small molecule anticancer drugs(e.g.doxorubicin,camptothecin etc.)and biological macromolecules(e.g.plasmids,siRNA and protein etc.).Compared with other drug vectors,CPPs are characterized by biocompatibility,biodegradability,low toxicity,low cost and high delivery efficiency.The cellular uptake mechanism of CPPs is not yet fully understood.At present,the main uptake mechanisms are energy-independent direct membrane translocation and energy-dependent endocytosis.A majority of CPPs enters cell by endocytosis.The cellular uptake mechanism of CPPs is complicated.Because different types of CPPs enter cell through different uptake mechanism,and the same CPP is affected by concentration and cell type in cellular uptake,so it often arises some contradictory conclusions.CPPs are positively charged due to the cationic amino acid residues in the sequences.They are adsorbed on the cell surface by electrostatic interaction,and then enter cell,but the detail of the uptake process is currently unknown.Heparan sulfate(HS)is a negatively charged biopolymer on cell surface which composed of different sulfated repeating disaccharide units.It is connected to membrane protein by covalent bond to form heparan sulfate proteoglycan(HSPG).HSPG is involved in regulating the metabolism,proliferation and angiogenesis in tumor cells.And a number of studies have shown that the cellular uptake of CPPs is also related to HSPG.CyLoP-1 was originally derived from Crotamine.It consists of 10 amino acids,and is rich in cationic residues.Compared with other known CPPs,it is characterized by high cellular uptake efficiency and low cytotoxicity under low concentration.More importantly the uptake of CyLoP-1 is strongly dependent on cell type,but there is no explanation on the cell selectivity.This study focused on the cellular uptake mechanism of the CyLoP-1.The results showed that CyLoP-1 has high uptake efficiency in Hela and A549(model cell line with high expression of heparan sulfate),and almost no uptake in Jurkat(model cell line with low expression of heparan sulfate),which indicating that the uptake of CyLoP-1 was related to heparan sulfate on the membrane.It also explained that its cell selectivity was related to the expression of heparan sulfate on cell surface.A series of CyLoP-1 analogs were designed to explore the structure-activity relationship.The result indicated that CyLoP-M1 and CyLoP-M4 showed higher transmembrane activity than CyLoP-1.After cytotoxicity test,lactate dehydrogenase release test,and hemolytic activity test,the results showed that the cytotoxicity of the modified analogues did not increase,and retained the characteristic of low toxicity.The subsequent isothermal titration calorimetry(ITC)and dynamic light scattering(DLS)experiment showed that the HS binding ability of CyLoP-M1 and CyLoP-M4 is higher than CyLoP-1,and analogues can induce more heparan sulfate to cluster.Considering the previous cellular uptake experiments,a reasonable hypothesis is proposed to explain why the transmembrane activity of CyLoP-M1 improved.CPPs binding to heparan sulfate on the membrane and inducing heparan sulfate to cluster are crucial in the process of cellular uptake,which promote the cell endocytosis.The phospholipid peptide cannot pass through the cell membrane due to its electronegativity,so it can be used to evaluate the cargo delivery capacity of the CPPs.Phospholipid peptide was connected to N-terminus of CyLoP-M1 covalently.The result showed that not only phospholipid peptide was delivered into cell successfully,but also the delivery efficiency was higher than CyLoP-1.Therefore,CyLoP-M1 has the potential to become a novel CPP. |
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