Ritu Goyal1, Chintan H Kapadia1, Jilian R Melamed1, Rachel S Riley1, Emily S Day1,2,3. 1. Department of Biomedical Engineering, University of Delaware, 161 Colburn Lab, Newark, DE 19716 USA. 2. Department of Materials Science & Engineering, University of Delaware, Newark, DE USA. 3. Helen F. Graham Cancer Center & Research Institute, Newark, DE USA.
Abstract
INTRODUCTION: MicroRNAs (miRNAs) are short noncoding RNAs whose ability to regulate the expression of multiple genes makes them potentially exciting tools to treat disease. Unfortunately, miRNAs cannot passively enter cells due to their hydrophilicity and negative charge. Here, we report the development of layer-by-layer assembled nanoshells (LbL-NS) as vehicles for efficient intracellular miRNA delivery. Specifically, we developed LbL-NS to deliver the tumor suppressor miR-34a into triple-negative breast cancer (TNBC) cells, and demonstrate that these constructs can safely and effectively regulate the expression of SIRT1 and Bcl-2, two known targets of miR-34a, to decrease cell proliferation. METHODS: LbL-NS were made by coating negatively charged nanoshells with alternating layers of positive poly-L-lysine (PLL) and negative miRNA, with the outer layer consisting of PLL to facilitate cellular entry and protect the miRNA. Electron microscopy, spectrophotometry, dynamic light scattering, and miRNA release studies were used to characterize LbL-NS. The particles' ability to enter MDA-MB-231 TNBC cells, inhibit SIRT1 and Bcl-2 expression, and thereby reduce cell proliferation was examined by confocal microscopy, Western blotting, and EdU assays, respectively. RESULTS: Each successive coating reversed the nanoparticles' charge and increased their hydrodynamic diameter, resulting in a final diameter of 208±4 nm and a zeta potential of 53±5 mV. The LbL-NS released ~30% of their miR-34a cargo over 5 days in 1X PBS. Excitingly, LbL-NS carrying miR-34a suppressed SIRT1 and Bcl-2 by 46±3% and 35±3%, respectively, and decreased cell proliferation by 33%. LbL-NS carrying scrambled miRNA did not yield these effects. CONCLUSION: LbL-NS can efficiently deliver miR-34a to TNBC cells to suppress cancer cell growth, warranting their further investigation as tools for miRNA replacement therapy.
INTRODUCTION: MicroRNAs (miRNAs) are short noncoding RNAs whose ability to regulate the expression of multiple genes makes them potentially exciting tools to treat disease. Unfortunately, miRNAs cannot passively enter cells due to their hydrophilicity and negative charge. Here, we report the development of layer-by-layer assembled nanoshells (LbL-NS) as vehicles for efficient intracellular miRNA delivery. Specifically, we developed LbL-NS to deliver the tumor suppressor miR-34a into triple-negative breast cancer (TNBC) cells, and demonstrate that these constructs can safely and effectively regulate the expression of SIRT1 and Bcl-2, two known targets of miR-34a, to decrease cell proliferation. METHODS: LbL-NS were made by coating negatively charged nanoshells with alternating layers of positive poly-L-lysine (PLL) and negative miRNA, with the outer layer consisting of PLL to facilitate cellular entry and protect the miRNA. Electron microscopy, spectrophotometry, dynamic light scattering, and miRNA release studies were used to characterize LbL-NS. The particles' ability to enter MDA-MB-231 TNBC cells, inhibit SIRT1 and Bcl-2 expression, and thereby reduce cell proliferation was examined by confocal microscopy, Western blotting, and EdU assays, respectively. RESULTS: Each successive coating reversed the nanoparticles' charge and increased their hydrodynamic diameter, resulting in a final diameter of 208±4 nm and a zeta potential of 53±5 mV. The LbL-NS released ~30% of their miR-34a cargo over 5 days in 1X PBS. Excitingly, LbL-NS carrying miR-34a suppressed SIRT1 and Bcl-2 by 46±3% and 35±3%, respectively, and decreased cell proliferation by 33%. LbL-NS carrying scrambled miRNA did not yield these effects. CONCLUSION: LbL-NS can efficiently deliver miR-34a to TNBC cells to suppress cancer cell growth, warranting their further investigation as tools for miRNA replacement therapy.
Entities:
Keywords:
Bcl-2; RNA interference; SIRT-1; gene regulation; microRNA; nanoparticles; poly-L-lysine; release; trafficking; triple-negative breast cancer
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