具有荧光探针的靶向肽/聚(醚-酰胺)制备及其肝癌细胞靶向性能研究

通过端氨基聚乙二醇PEG(Ⅰ)与二亚乙基三胺五乙酸二酐(DTPAA)开环合成新型端氨基聚(醚-酰胺)(PEG/DTPA)共聚物造影剂配体(Ⅱ)(Step1);Ⅱ的端氨基与偶联剂3-马来酰亚胺苯甲酸-N-琥珀酰亚胺酯(MBS)的活化端COOH反应,生成偶联剂/聚(醚-酰胺)MB/PEG/DTPA(Ⅲ′)(Step2);再通过Ⅲ′中MBS的CC双键与肝癌细胞靶向黏附肽FAM-AGKGTPSLETTPC-(SH)-COOH(FAM-13)上的巯基SH发生Michael加成反应(Step3),合成含有荧光探针FAM(5-carboxyfluorescein)的肝癌靶向肽/聚(醚-酰胺)(FAM-13/PEG/DTPA,Ⅲ).用1H-NMR和13C-NMR等方法对共聚物进行表征.Ⅲ对正常肝细胞L-02几乎观察不到荧光现象,而对肝癌细胞BEL-7404则有很强的黄绿色荧光,Ⅲ对肝癌细胞有很强的靶向性.大分子配体Ⅲ可望用于制备大分子造影剂及靶向载体负载药物.

SYNTHESIS OF FLUORESCENE LABELED PEPTIDE/POLY(ETHER-AMIDE)WITH TARGETING FUNCTION TO LIVER CANCER CELLS

To develop and characterize a new cancer target gene delivery carrier,which could be used as potential blood pool magnetic resonance ( MR ) contrast agents,a novel FAM-AGKGTPSLETTPC-COOH / poly(ether-amide)(FAM-13 /PEG /DTPA)copolymer (Ⅲ)with fluorescence probe was designed and obtained first from the reaction of the amino group terminated poly(ether-amide)(PEG /DTPA)copolymer ligand ( Ⅱ) with heterobifunctional cross-linker m-maleimidobenzoyl-N-hydroxysuccinimide ester ( MBS) ( Step 2 ),and then Michael addition of C C double bond in MBS moiety with SH group in FAM-labeled hepatocarcinoma specific adhesin peptide,FAM-AGKGTPSLETTPC-( SH)-COOH ( FAM-13),in high yield ( Step 3).Ⅱ was prepared by the reaction of diethylenetriamine pentaacetic acid dianhydrid ( DTPAA ) with amino group terminated PEG(Ⅰ)(Step 1).The two copolymers of Ⅱ and Ⅲ were characterized by 1H-NMR and 13C-NMR measurements.The liver tumor-specific identification and antigen localization of Ⅲ with the fluorescin-labeled FAM-13 peptide were performed under cellular level.Targeting properties of Ⅲ were tested on the normal cells and liver cancer cells,respectively.No fluorescence was observed on normal liver cells L-02,while strong yellowish green fluorescence was observed on the liver cancer cells BEL-7404.The property of FAM-13 peptide in Ⅲ for recognizing specifically the liver cancer cells was demonstrated.It was also demonstrated that the chemically synthesized Ⅲ with peptide FAM-13 still had the property for targeting on the surface membrane of liver cancer cells in vitro,thus further indicating that the specific targeting effect of the objective tissue was mediated by the peptides of Ⅲ insert into the cell membrane.It is evident that the Ⅲ with FAM-13 peptide would be an excellent candidate for the targeting therapy to hepatocarcinoma.So ligand molecules Ⅲ with targeting peptides can target hepatocellular carcinoma.Ligand Ⅲ is a promising candidate,which can be coordinated with Gd3 + ion to give the macromolecular contrast agent.