Layer-by-Layer Assembled Gold Nanoshells for the Intracellular Delivery of miR-34a

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.

Layer‐by‐layer assembled PLGA nanoparticles carrying miR‐34a cargo inhibit the proliferation and cell cycle progression of triple‐negative breast cancer cells

“…Delivering mimics of the tumor suppressor miR-34a, whose expression is lost in TNBC, may improve patient prognosis (Saito et al, 2015). LbL NPs formed by sequential deposition of alternating polyelectrolyte layers around a spherical core show great promise as miRNA and siRNA delivery vehicles that can overcome several of these delivery barriers (Choi et al, 2015;Deng et al, 2013;Elbakry et al, 2009;Goyal et al, 2018;Gu et al, 2017). However, effective miR-34a delivery is incredibly challenging to achieve because there are a number of biological barriers that miRNA carriers must overcome.…”

“…First, PLL is a pH-sensitive biodegradable polycation that holds positive charge at physiological pH to facilitate electrostatic binding of negatively charged miRNAs (Choi et al, 2015). Moreover, PLL facilitates endosomal escape of RNA cargo for successful delivery into the cell cytosol (Choi et al, 2015;Deng et al, 2013;Goyal et al, 2018). Moreover, PLL facilitates endosomal escape of RNA cargo for successful delivery into the cell cytosol (Choi et al, 2015;Deng et al, 2013;Goyal et al, 2018).…”

“…Previously Hammond and colleagues synthesized LbL NPs in which miR-34a and KRAS siRNA were sandwiched between poly-L-arginine layers surrounding a cisplatin-loaded liposomal core, and showed that these were effective against lung cancer in both cell culture and rodent models (Gu et al, 2017). Extending this strategy to TNBC, we have shown that miR-34a can be delivered to TNBC cells using LbL NPs prepared with silica core/gold shell nanoshells as the NP scaffold and poly-L-lysine (PLL) as the polycation (Goyal, Kapadia, Melamed, Riley, & Day, 2018). One disadvantage of this system is that the nanoshell core is not biodegradable, and will remain in the body for extended periods of time.…”

Polymer nanocarriers for MicroRNA delivery

“…With respect to size, nanoscale materials are ideally suited for RNA delivery because they can protect RNA from degradation, extend circulation half‐life, facilitate cellular entry, and increase therapeutic index . Indeed, various nanoparticle (NP) delivery systems have shown considerable promise for the delivery of plasmid DNAs, antisense oligonucleotides, small interfering RNAs (siRNAs), and miRNAs to diseased cells in vitro and in vivo . When considering size as a design parameter for miRNA mimic delivery vehicles, it is important to note that NPs with diameters less than ∼5 nm rapidly undergo renal clearance upon intravenous administration and those with diameters greater than ~200 nm exhibit splenic filtration due to the 200–500 nm size range of interendothelial cell slits .…”

“…Composition will dictate whether the miRNA is loaded on the surface of the carrier or encapsulated within the carrier, and it will also dictate the degradation profile (if any), as well as the rate of cargo release. Metal‐based NPs, liposomes, polymer nanocarriers (including polymer NPs, polyplexes, and dendrimers), lipid NPs, hydrogels, and more have been explored for the delivery of siRNA and miRNA. The benefits and limitations of lipid NPs and liposomes and of metal NPs as miRNA delivery vehicles have been reviewed elsewhere .…”