低相对分子质量肝素接枝羧甲基纤维素超支化聚合物的合成、表征及活性研究

Synthesis,Characterization and Biological Activity of a LMWH Hyperbranched Carboxymethylcellulose Polymer

为提高低分子量肝素的生物活性,用末端接枝法将末端醛基低相对分子质量肝素(LMWH-CHO)缀合在羧甲基纤维素(CMC)主链上,形成一种强负电性的超支化聚合物(LMWH-grafted CMC,CH)。LMWH-CHO是通过亚硝酸钠降解法制备获得,利用EDC催化酰胺键生成反应使CMC氨基化,然后通过醛基和氨基的希夫碱反应将二者缀合。运用傅立叶变换红外光谱(FTIR)、核磁共振(NMR)、凝胶渗透色谱-多角度激光散射联用技术(GPC-MALLS)等方法对产物结构和分子量进行表征,并对聚合物的抗凝血、抗血栓活性进行了测定。结果显示,在氨基化CMC糖单元与LMWH-CHO摩尔比为1∶5的条件下反应12 h,得到接枝率为23.7%的CH超支化聚合物。该聚合物活化部分凝血活酶时间(APTT)为146 s,比LMWH延长了25%,凝血酶原时间(PT)为27.9 s,约为LMWH的2倍。此外,该聚合物抗Xa IC50值为104.2 nmol/L,比LMWH降低了近4倍;抗IIa IC50值为642 nmol/L,远低于LMWH(>2 000 nmol/L)。表明,制备的CH超支化聚合物抗凝血和抗血栓活性均远优于低相对分子质量肝素。

A strongly negative hyperbranched copolymer （LMWH-grafled CMC,CH） was synthesized by low-molecular-weight heparin （LMWH） wafted onto carboxymethylcellulose （CMC） backbone to improve the biological activities of LMWH via multivalent effect. LMWH with an aldehyde group at their reduce ends （LMWH-CHO） was prepared from heparin degradation by nitrous acid. The amide bond was formed between the amino group of EDA and the carboxylic groups of CMC when catalyzed by EDC and the primary amino groups were introduced to the backbone （CMC-NH2）. LMWH-CHO was thus grafted onto CMC-NH2 by the Schiff-base reaction.The synthesized hyperbranched polymer was characterized by FTIR,NMR and GPC-MALLS to detect its chemical structure and molecular weight. Furthermore ,the anticoagulation activities of the polymer were determined. The results showed that the grafted ratio of CMC was 23.7% with a molar ratio of CMC-NH2 to LMWH-CHO of 1：5. The APTT and PT of the polymer were 146 s and 27.9 s, respectively,which were 1.25-fold and 2-fold higher compared with that of LMWH. In addition,the IC50 value of anti-factor Xa and anti-factor IIa of the polymer were 104.2 nmol/L and 642 nmol/L, respectively. The values were much lower than those of LMWH. In conclusion, LMWH grafted CMC polymer possessed a better anticoagulation activity than LMWH.