ELUCIDATION OF THE MECHANISM OF EFFECTIVE MEMBRANE PERMEATION OF BUFORIN 2 AND ITS APPLICATION TO VECTOR FOR INTRACELLULAR DRUG DELIVERY

从包括人和植物在内的各种动物中已经发现了500多种抗菌肽。这些肽现在被认为在天然免疫中起重要作用。许多抗菌肽，如从非洲爪蟾中分离的马盖宁2，靶向骨髓细胞膜，通过渗透细胞膜而产生细胞毒性。从中华大蟾蜍（Bufo BUFO GARGARIZANS）中分离的蟾蜍素2（Buforin 2）能不经膜渗透而通过结合DNA和RNA杀死芽孢杆菌。本研究的目的是：1）阐明蟾蜍素2的连续易位机制; 2）将这种独特的性质应用于细胞内药物递送。1）易位机制：蟾毒蛋白2通过与马盖宁2基本相同的机制，即通过形成瞬时肽-脂质超分子复合物孔，在脂质双层上移位。由脯氨酸11的存在形成的短两肢螺旋（残基5-21）含有许多正电荷。因此，静电排斥极大地使孔失稳，使膜渗透最小化。2）细胞内药物递送：在N-或C-血管中，将具有德克萨斯红染料的蟾毒灵2衍生物作为药物模型，并测定其与人细胞系（HeLa和TM 12）的相互作用。这些肽即使在存在代谢抑制剂的情况下也不依赖于代谢酶而穿透细胞，此外，这些肽的毒性非常低。因此，Buforin 2是细胞内药物递送载体的有希望的候选者。最后，本研究的目的可以在该资助的帮助下实现。

MORE THAN 500 ANTIMICROBIAL PEPTIDES HAVE BEEN DISCOVERED FROM VARIOUS ANIMALS INCLUDING HUMAN AND PLANTS. THESE PEPTIDES ARE NOW RECOGNIZED TO PLAY AN IMPORTANT ROLE IN INNATE IMMUNITY. MANY ANTIMICROBIAL PEPTIDES, LIKE MAGAININ 2 ISOLATED FROM XENOPUS LAEVIS, TARGET BACTERIAL CELL MEMBRANES, EXERTING CYTOTOXICITY BY PERMEABILIZING THEM. IN CONTRAST, BUFORIN 2 ISOLATED FROM BUFO BUFO GARGARIZANS EFFECTIVELY CROSS MEMBRANES WITHOUT PERMEABILIZATION, KILLING BACTERIA BY BINDING DNA AND RNA.THE AIMS OF THIS STUDY ARE 1)TO ELUCIDATE THE MECHANISM OF THE EFFECTIVE TRANSLOCATION OF BUFORIN 2 AND 2)TO APPLY THIS UNIQUE PROPERTY TO INTRACELLULAR DRUG DELIVERY.1)MECHANISM OF TRANSLOCATION : BUFORIN 2 TRANSLOCATES ACROSS LIPID BILAYERS BY A MECHANISM ESSENTIALLY THE SAME AS THAT OF MAGAININ 2, I.E. VIA THE FORMATION OF A TRANSIENT PEPTIDE-LIPID SUPRAMOLECULAR COMPLEX PORE. THE SHORT AMPHIPATHIC HELIX (RESIDUES 5-21) FORMED BY THE PRESENCE OF PROLINE11 CONTAINS MANY POSITIVE CHARGES. THEREFORE, THE ELECTROSTATIC REPULSION EXTREMELY DESTABILIZES THE PORE, MINIMIZING MEMBRANE PERMEABILIZATION.2)INTRACELLULAR DRUG DELIVERY : BUFORIN 2 DERIVATIVES WITH THE TEXAS RED DYE AS A DRUG MODEL AT THE N- OR C-TERMINUS WERE SYNTHESIZED, AND THEIR INTERACTIONS WITH HUMAN CELL LINES (HELA AND TM12) WERE INVESTIGATED. THESE PEPTIDES TEMPERATURE-INDEPENDENTLY PENETRATED CELLS EVEN IN THE PRESENCE OF A METABOLIC INHIBITOR, FURTHERMORE, THE TOXICITIES OF THESE PEPTIDES WERE VERY LOW. THUS, BUFORIN 2 IS A PROMISING CANDIDATE OF A VECTOR FOR INTRACELLULAR DRUG DELIVERY.FINALLY, THE AIMS OF THIS STUDY COULD BE ACHIEVED BY THE AID OF THIS GRANT.