Transferrin Receptor-Targeted Liposomes Encapsulating anti-BCR-ABL siRNA or asODN for Chronic Myeloid Leukemia Treatment
Transferrin Receptor-Targeted Liposomes Encapsulating anti-BCR-ABL siRNA or asODN for Chronic Myeloid Leukemia Treatment
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DOI：
10.1021/bc9004365
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发表时间：
2010-01-01
影响因子：
4.7
通讯作者：
Simoes, Sergio
中科院分区：
文献类型：
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作者：
Mendonca, Liliana S.;Firmino, Filipe;Simoes, Sergio
本工作旨在开发和应用以转铁蛋白受体(TrfR)为靶点的空间稳定的脂质体，包裹抗bcr-abl siRNA或asODN。转铁蛋白通过后插入的方法偶联到包裹siRNA或asODN的脂质体表面。用流式细胞仪和共聚焦显微镜分别检测细胞的结合和内化。采用定量逆转录聚合酶链式反应(qRT-PCR)和免疫印迹法检测bcr-abl mRNA和蛋白表达水平。细胞活性测定采用雷氏天青还原法测细胞活性。偶联转铁蛋白的量、脂质体的大小和随时间的稳定性都非常令人满意，并且重复性好。SiRNA包封率取决于所使用的包封缓冲液的浓度(20或300 mM)，而asODN包封率在所测试的两种浓度下都很高。用TRF(TRF-脂质体)或白蛋白(BSA-脂质体)偶联的脂质体(BSA-脂质体)或包裹荧光标记的siRNA(Cy3-siRNA)的非靶向脂质体(NT-脂质体)处理白血病细胞系，进行细胞结合和内化研究。这些实验清楚地表明，BSA和NT脂质体不能促进包裹的核酸的传递，而TRF脂质体是通过TRF受体依赖的机制传递核酸的。包裹siRNA或asODN的TRF脂质体促进序列特异性的bcr-abl mRNA下调，尽管在蛋白质和细胞活力水平观察到一定程度的非特异性序列效应。总体而言，我们的结果表明，TRF脂质体包裹的基因沉默工具允许结合分子和细胞靶向，这是一种有价值的癌症治疗方法。
The present work aimed at the development and application of transferrin receptor (TrfR)-targeted sterically stabilized liposomes encapsulating anti-BCR-ABL siRNA or asODN. Transferrin Was Coupled to the surface of liposomes encapsulating siRNA or asODN through the postinsertion method. Cell association and internalization were assessed by flow cytometry and confocal microscopy, respectively. BCR-ABL mRNA and Bcr-Abl protein levels were evaluated by qRT-PCR and Western blot, respectively. Cell viability was assessed using the resazurin reduction method. The amount Of coupled transferrin and the size and stability over time of the liposomes were very satisfactory and reproducible. The siRNA encapsulation yield was dependent on the concentration of the encapsulation buffer used (20 or 300 mM), as opposed to asODN encapsulation yield which was high for both concentrations tested. Cell association and internalization studies were performed in leukemia cell lines treated with liposomes coupled to Trf (Trf-liposomes) or albumin (BSA-liposomes) or with nontargeted liposomes (NT-liposomes) encapsulating fluorescently labeled siRNA (Cy3-siRNA). These experiment, clearly indicated that BSA- and NT-liposomes have no ability to promote the delivery of the encapsulated nucleic acids and that the Trf-liposomes deliver the nucleic acids by it Trf receptor-dependent mechanism. The Trf-liposomes encapsulating siRNA or asODN promote sequence-specific down-regulation of the BCR-ABL mRNA, although a certain extent of nonspecific sequence effects at the protein and cell viability level were observed. Overall, our results indicate that Trf-liposomes encapsulating gene silencing tools allow combining molecular and cellular targeting, which is it valuable approach for cancer treatment.