多柔比星PEG_(2K)-(Fmoc)lys-NLG_(919)胶束的动物心肌毒性实验

Cardiac toxicity of doxorubicin loaded PEG_(2K)-(Fmoc)lys-NLG_(919) micelle:in vitro and in vivo evaluation experiment

目的研究聚乙二醇2000-芴酰-NLG_(919)(PEG_(2K)-(Fmoc)lys-NLG_(919))载体及形成多柔比星胶束后其体内和体外心毒性。方法获取并培养大鼠心肌细胞,MTT法检测相同浓度梯度下盐酸多柔比星、空白胶束载体和DOX胶束对大鼠心肌细胞增殖影响的差异;将小鼠随机分为空白对照组(n=5)、DOX组(n=5)、空白胶束载体组(n=5)和DOX胶束组(n=5),每组静脉注射3次相应药物后分析血清肌酸激酶(creatine kinase,CK)、肌酸激酶同工酶(creatine kinase isoenzyme,CK-MB)、乳酸脱氢酶(lactate dehydrogenase,LDH)浓度,比较差异并取小鼠心脏进行HE染色镜下观察。结果体外MTT示空白载体各浓度细胞活性无统计学差异(P>0.05),DOX胶束给药组细胞活性明显高于盐酸DOX组(DOX 1 ng/ml时盐酸DOX组细胞活性53.12%±6.84%,DOX胶束组细胞活性74.48%±6.16%,P<0.05)。体内实验空白胶束组[CK(333.73±51.60)U/L、CK-MB(43.07±2.17)U/L、LDH(657.33±23.94)U/L]和DOX胶束组[CK(538.27±48.71)U/L,CK-MB(53.60±2.10)U/L,LDH(857.33±16.46)U/L]明显低于盐酸DOX组[CK(1 307.67±81.64)U/L,CK-MB(151.07±12.33)U/L,LDH(2610.67±21.39)U/L,P<0.05]。HE染色病理切片示盐酸DOX组心肌破坏严重,空白胶束载体组无明显心肌破坏,DOX胶束组心肌无明显破坏。结论 PEG_(2K)-(Fmoc)lys-NLG_(919)胶束载体本身无心肌毒性,且包裹DOX后可明显降低DOX毒性。

Objective To explore the cardiac toxicity of doxorubicin loaded PEG2K-（Fmoc）lys-NLG919＇s micelle and drug free micelle in vitro and in vivo.Methods For in vitro study, newborn rat＇s mycardial cells are treated with a seriers concentrations of free doxorubicin, free carrier and doxorubicin loaded micelle, and the differences of cell viabilities were evaluated by MTT assay.For in vivo study, 20 mice are randomly divided into four groups：control group（n=5）, free DOX group（n=5）, free carrier group（n=5）, DOX loaded micelle group（n=5）.Mice in four groups are treated by 3 times injection of Saline, free doxorubicin, free carrier and doxorubicin loaded micelle, respectively.After last injection, serum were tested for the level of CK,CK-MB and LDH.All the mice were sacrificed and their hearts were examined by HE staining to observe the changes of mycardial cells by microscope.Results For in vitro study, drug free micelle with different concentrations have no cardiac toxicity（P〈0.05）.The cell viability of the DOX loaded micelles group（DOX 1ng/ml） was significantly higher than that of free DOX group（DOX 1 ng/ml）（74.48%±6.16% vs 53.12%±6.84%,P〈0.05）.For in vivo study, serum levels of CK, CK-MB, LDH in free carrier group（（333.73±51.60）U/L,（43.07±2.17） U/L,（657.33±23.94） U/L）and in DOX loaded micelle group [CK（538.27±48.71）U/L, CK-MB（53.60±2.10）U/L, LDH（857.33±16.46） U/L）] were significantly lower than those in free DOX group（CK（1 307.67± 81.64）U/L; CK-MB（151.07±12.33） U/L; LDH（2610.67±21.39）U/L.P〈0.05, respectively）.The pathological changes in HE staining showed the DOX induced myocardial damage seriously, but no obvious myocardial damage was observed in the free carrier and DOX loaded micelle group.Conclusion The drug free PEG2K-（Fmoc）lys-NLG919 carrier have no cardiac toxicity.Doxorubicin loaded PEG2K-（Fmoc） lys-NLG919 micelle can obviously reduce the cardiac toxicity from doxorubicin.