纳米载体靶向共转运GLUT1-siRNA和羟基喜树碱逆转急性髓系白血病化疗耐受的研究

The mechanism of multidrug resistance (MDR) in acute myeloid leukemia (AML) is complex and the effect of single drug treatment is not ideal. Treating MDR thus requires multipronged approaches such as co-administration of drug/gene combinthe ttherapeutic efficacy ofation treatments using targeted nanoparticles. Our preliminary study showed that high glucose metabolism mediated by overexpression of glucose transporter 1 (GLUT1) existed in drug-resistant AML cells, and down-regulation of the expression of GLUT1 by RNA interference obviously sensitized drug-resistant HL-60/ADR cells to adriamycin (ADR) in vitro. Targeting for folate (FA) receptor of leukemia cell, 10-Hydroxy camptothecin (HCPT), a chemotherapeutic drug, and FA were grafted polyethyleneimine (PEI). Meanwhile the PEI cationic nanocarrier allowed for electrostatic interaction with the negatively charged small interfering RNA (siRNA)-targeting GLUT1 gene (siGLUT1). The biological effects of the new complex micelles will be investigated in vitro and in vivo, and their active targeting ability, regulation of glucose metabolism, associated molecular expression of MDR and apoptosis were analyzed for MDR treatment. The data may provide a novel strategy of targeted co-delievery for chemoresistance reversal in AML.

急性髓系白血病(AML) 多药耐药（MDR）的机制复杂,单一药物治疗效果并不理想。纳米载体可靶向介导siRNA和药物共转运已受到关注并应用于MDR的治疗。前期研究发现耐药AML细胞存在葡萄糖转运蛋白1(GLUT1)过表达介导的高葡萄糖代谢，GLUT1基因沉默可增强耐药细胞的化疗敏感性。由于AML细胞高表达叶酸（FA）受体，通过FA接枝阳离子聚合物聚乙烯亚胺（PEI）合成主动靶向纳米载体，并以酯键连接化疗药羟基喜树碱（HCPT），通过PEI的正电荷吸附GLUT1-siRNA（siGLUT1）合成并新型材料siGLUT1-HCPT-PEI-FA。新合成材料通过FA的主动靶向性、siGLUT1联合HCPT的共转运体内外作用于耐药AML细胞，评价其生物学效应；通过分析其主动靶向性、糖代谢能量调控、MDR和凋亡分子表达等阐明其逆转AML化疗耐受的作用机理，为逆转AML的化疗耐受性提供新的思路。

急性髓系白血病(AML) 多药耐药（MDR）的机制复杂，故针对 MDR 的治疗可能需要多种不同作用机制的药物或基因联合应用来发挥协同抗肿瘤作用。纳米载体可靶向介导siRNA和药物共转运已受到关注并应用于MDR的治疗。既往我们的研究发现耐药AML细胞存在葡萄糖转运蛋白1(GLUT1)过表达介导的高糖酵解代谢表型，GLUT1基因沉默可增强耐药AML细胞的化疗敏感性。另外，结合AML细胞高表达透明质酸(HA)受体，我们通过合成HA靶向的共转运GLUT1-siRNA和HCPT的Au-SBC-HA纳米材料，从而评价其生物学效应。首先，通过原位还原加静电吸附将金纳米颗粒（Au）结合到支链化壳聚糖（SBC）上，然后以Au为模板，通过亲疏水反向结晶的方式将HCPT层积于Au-SBC表面。最后将siGLUT1和HA以静电吸附方式结合到Au-SBC-HCPT上形成HA靶向的多功能新型纳米材料Au-SBC-HA-siGLUT1-HCPT。结果提示，新型材料Au-SBC-HA-siGLUT1-HCPT不仅解决了化疗药物HCPT溶解性差的问题，更具有较为理想粒径(179.9 nm)、zeta电位(-1.3 mV), 以及载药量（28.6%）。琼脂糖凝胶电泳结果表明siGLUT1确实结合到纳米材料上，高效液相质谱（HCPT）实验证实了材料良好缓释性能。荧光显微镜结果提示，结合HA的靶向材料明显提高了细胞对HCPT的摄取作用，尤其是在耐药细胞株HL-60/HCPT中。同样与HL-60细胞相比，共转运材料明显抑制了耐药细胞HL-60/HCPT的增殖活性和葡萄糖消耗。综上，共转运siGLUT1和HCPT的HA靶向新型纳米胶束Au-SBC-HA-siGLUT1-HCPT可通过其主动靶向和下调糖酵解活性等多重作用来提高化疗敏感性，为逆转AML的MDR提供了新的靶向载药纳米体系和思路。