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Hongyan Zhou,1,* Jinhuan Wei,2,* Qiangsheng Dai,3 Liping Wang,4 Junhang Luo,2 Tuckyun Cheang,4 Shenming Wang4 1Department of Neurological Intensive Care Unit, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People’s Republic of China; 2Department of Urology, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People’s Republic of China; 3Department of Oncology, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People’s Republic of China; 4Department of Breast Surgery, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People’sRepublic of China *These authors contributed equally tothis work Background: Small interfering RNA (siRNA)-mediated gene therapy is a promising strategy to temporarily inhibit the expression of genes involved in development of breast cancer. The lack of a safe and efficient gene delivery system has become a major hurdle for siRNA-mediated gene therapy in breast cancer. Our previous studies have demonstrated that inorganic amorphous calcium carbonate (ACC) hybrid nanospheres functionalized with CaIP6 (ACC/CaIP6) nanoparticles are an efficient nucleic acid delivery tool. The present study aimed to evaluate the safety and efficiency of ACC/CaIP6 in delivering siRNA targeting AKT1 (siAKT1) for the treatment of breast cancer. Methods: The cytotoxicity of the ACC/CaIP6 nanoparticles was evaluated using a tetrazolium assay. The transfection efficiency and intracellular distribution of ACC/siAKT1 were analyzed by flow cytometry and confocal laser scanning microscopy, respectively. A series of in vitro and in vivo assays was performed to evaluate the effects of ACC/CaIP6/siAKT1 on growth of breast cancer cells. Results: ACC/CaIP6 nanoparticles effectively transfected cells with little or no toxicity. AKT1 knockdown by ACC/CaIP6/siAKT1 inhibited cell cycle progression and promoted apoptosis of MCF-7 cells. Intratumoral injection of ACC/CaIP6/siAKT1 significantly suppressed the growth of breast cancer in mice. Conclusion: ACC/CaIP6 nanoparticles are a safe and efficient method of delivering siRNA for gene therapy in breast cancer. Keywords: breast cancer, gene therapy, nanoparticles, small interfering RNA

Guanhua Li,1,2 Zuojun Hu,1 Henghui Yin,1 Yunjian Zhang,1 Xueling Huang,1 Shenming Wang,1 Wen Li2 1Department of Vascular and Thyroid Surgery, 2Key Laboratory of Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, People's Republic of China Abstract: The application of RNA interference techniques is promising in gene therapeutic approaches, especially for cancers. To improve safety and efficiency of small interfering RNA (siRNA) delivery, a triblock dendritic nanocarrier, polyamidoamine-polyethylene glycol-cyclic RGD (PAMAM-PEG-cRGD), was developed and studied as an siRNA vector targeting the human ether-à-go-go-related gene (hERG) in human anaplastic thyroid carcinoma cells. Structure characterization, particle size, zeta potential, and gel retardation assay confirmed that complete triblock components were successfully synthesized with effective binding capacity of siRNA in this triblock nanocarrier. Cytotoxicity data indicated that conjugation of PEG significantly alleviated cytotoxicity when compared with unmodified PAMAM. PAMAM-PEG-cRGD exerted potent siRNA cellular internalization in which transfection efficiency measured by flow cytometry was up to 68% when the charge ratio (N/P ratio) was 3.5. Ligand-receptor affinity together with electrostatic interaction should be involved in the nano-siRNA endocytosis mechanism and we then proved that attachment of cRGD enhanced cellular uptake via RGD-integrin recognition. Gene silencing was evaluated by reverse transcription polymerase chain reaction and PAMAM-PEG-cRGD-siRNA complex downregulated the expression of hERG to 26.3% of the control value. Furthermore, gene knockdown of hERG elicited growth suppression as well as activated apoptosis by means of abolishing vascular endothelial growth factor secretion and triggering caspase-3 cascade in anaplastic thyroid carcinoma cells. Our study demonstrates that this novel triblock polymer, PAMAM-PEG-cRGD, exhibits negligible cytotoxicity, effective transfection, “smart” cancer targeting, and therefore is a promising siRNA nanocarrier. Keywords: small interfering RNA, dendrimer, gene silencing, human ether-à-go-go-related gene, anaplastic thyroid cancer

Tuck-yun Cheang1,*, Bing Tang1,*, An-wu Xu2, Guang-qi Chang1, Zuo-jun Hu1, Wei-ling He1, Zhou-hao Xing2, Jian-bo Xu1, Mian Wang1, Shen-ming Wang11Department of Vascular Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; 2Division of Nanomaterials and Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China *Both authors contributed equally to this workAbstract: Nanoparticles have an enormous potential for development in biomedical applications, such as gene or drug delivery. We developed and characterized aminopropyltriethoxysilane-functionalized silicon dioxide nanoparticles (APTES-SiNPs) for gene therapy. Lipofectamine® 2000, a commonly used agent, served as a contrast. We showed that APTES-SiNPs had a gene transfection efficiency almost equal to that of Lipofectamine 2000, but with lower cytotoxicity. Thus, these novel APTES-SiNPs can achieve highly efficient transfection of plasmid DNA, and to some extent reduce cytotoxicity, which might overcome the critical drawbacks in vivo of conventional carriers, such as viral vectors, organic polymers, and liposomes, and seem to be a promising nonviral gene therapy vector.Keywords: aminopropyltriethoxysilane, silicon dioxide nanoparticles, Lipofectamine® 2000, gene therapy vector, nanomedicine