Layer-by-Layer Assembled Gold Nanoshells for the Intracellular Delivery of miR-34a
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.
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.
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 1× 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.
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.
KeywordsMicroRNA Nanoparticles RNA interference Gene regulation Poly-l-lysine Trafficking Release SIRT1 Bcl-2 Triple-negative breast cancer
Dynamic light scattering
Dulbecco’s Modified Eagle Medium
Fetal bovine serum
Lysosomal-associated membrane protein 1
Layer-by-layer assembled nanoshells
Mander’s colocalization coefficient
Rotations per minute
Scanning electron microscopy
Nucleophilic aromatic substitution reaction
Triple-negative breast cancer
Ultra violet–visible spectrophotometry
This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under Award Number R35GM119659 (PI:Day). JRM received support from the Department of Defense through a National Defense Science and Engineering Graduate Fellowship. The content is solely the responsibility of the authors and does not necessarily represent the views of the funding agencies. The LSM880 confocal microscope was acquired with a shared instrumentation Grant (S10 OD016361) and access was supported by the NIH-NIGMS (P20 GM103446), the NSF (IIA-1301765), and the State of Delaware. The Hitachi S4700 used in this work was acquired with the Delaware INBRE Grant P20 GM103446.
All authors conceptualized the experiments. RG, CK, JM, and RR performed the experiments and analyzed the data. ED secured funding for the experiments. All authors wrote and revised the manuscript.
Conflict of interest
Ritu Goyal, Chintan Kapadia, Jilian Melamed, Rachel Riley, and Emily Day declare no conflicts of interest.
No animal or human studies were performed in this work.
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