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We described here the mechanisms by which small interfering RNA (siRNA) molecules incorporated in reconstituted high density lipoprotein (rHDL) were efficiently transferred into the cytoplasm of cells to perform target-specific therapy of tumor angiogenesis.Using fluorescent-tagged apolipoprotein A-I (apoA-I) and cholesterol-conjugated siRNA (Chol-siRNA),it was confirmed with FACS and confocal microscopic measurements that Chol-siRNA-loaded rHDL nanoparticles (rHDL/Chol-siRNA complexes) were successfully established and apoA-I certainly was attached to the surface of Chol-siRNA-loaded lipoplexes (Lipos/Chol-siRNA complexes).Stably assembled rHDL/Chol-siRNA complexes demonstrated proper nanosize,quasi-spherical shape and improved nuclease protection over naked Chol-siRNA.It was also interesting to note that rHDL provided a highly effective approach to transfer Chol-siRNA across the membrane directly into the cytoplasm via the scavenger receptor BI (SR-BI)-mediated non-endocytotic mechanism,thereby bypassing endo-lysosomal trapping.We also showed clear evidence that the in vitro implementation of rHDL for Chol-siRNA-VEGF (Chol-siRNA targeting vascular endothelial growth factor gene) delivery markedly promoted RNA interference (RNAi)-mediated degradation of VEGF mRNA,resulting in down-regulation of secreted VEGF protein.In vivo fluorescence imaging indicated that near-infrared (NIR) dye Cy5 labeled Chol-siRNA-loaded rHDL nanoparticles (rHDL/Cy5-Chol-siRNA complexes) displayed long circulation time,SR-BI positive tumor-selective targeting,and efficient cytosolic delivery capabilities.Furthermore,intravenous administration of Chol-siRNA-VEGF-loaded rHDL nanoparticles (rHDL/Chol-siRNA-VEGF complexes) significantly enhanced anti-tumor efficacy against breast cancer,decreased VEGF expression level,and inhibited formation of intratumoral microvessels at the tumor tissue.It was concluded that rHDL possessed therapeutic potential and versatility in mediating Chol-siRNA-VEGF direct cytosolic delivery for target-specific anti-angiogenic therapy in breast cancer.