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

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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 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.

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.

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Abbreviations

DEPC:

Diethyl pyrocarbonate

DLS:

Dynamic light scattering

DMEM:

Dulbecco’s Modified Eagle Medium

DMF:

Dimethylformamide

DNA:

Deoxyribonucleic acid

EdU:

5-Ethynyl-2′-deoxyuridine

FBS:

Fetal bovine serum

LAMP-1:

Lysosomal-associated membrane protein 1

LbL:

Layer-by-layer

LbL-NS:

Layer-by-layer assembled nanoshells

MCC:

Mander’s colocalization coefficient

mRNA:

Messenger RNA

miRNA:

microRNA

MUA:

11-Mercaptoundecanoic acid

NaCl:

Sodium chloride

NaOH:

Sodium hydroxide

NS:

Nanoshells

OD:

Optical density

PLL:

Poly-l-lysine

RIPA:

Radioimmunoprecipitation assay

RNA:

Ribonucleic acid

RNAi:

RNA interference

RPM:

Rotations per minute

SEM:

Scanning electron microscopy

SNAr:

Nucleophilic aromatic substitution reaction

TNBC:

Triple-negative breast cancer

TNBS:

2,4,6-Trinitrobenzenesulfonic acid

TNP:

Trinitrophenyl

TRITC:

Tetramethylrhodamine isothiocyanate

UV–Vis:

Ultra violet–visible spectrophotometry

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Acknowledgments

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.

Author contributions

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.

Ethical standards

No animal or human studies were performed in this work.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, University of Delaware, 161 Colburn Lab, Newark, DE, 19716, USA

    Ritu Goyal, Chintan H. Kapadia, Jilian R. Melamed, Rachel S. Riley & Emily S. Day

  2. Department of Materials Science & Engineering, University of Delaware, Newark, DE, USA

    Emily S. Day

  3. Helen F. Graham Cancer Center & Research Institute, Newark, DE, USA

    Emily S. Day

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  1. Ritu Goyal
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  2. Chintan H. Kapadia
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Correspondence to Emily S. Day.

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Associate Editor Lola Eniola-Adefeso oversaw the review of this article.

Emily S. Day is an Assistant Professor in the Department of Biomedical Engineering at the University of Delaware. Dr. Day completed her Ph.D. at Rice University in 2006, where she trained with Dr. Jennifer West. There, her research focused on developing nanoparticle-mediated photothermal therapy for treatment of glioblastoma, an aggressive form of primary brain tumor. While at Rice, Dr. Day received a National Science Foundation Graduate Research Fellowship, a Rice President’s Graduate Fellowship, and a Howard Hughes Medical Institute Med-Into-Grad Fellowship. Upon completing her Ph.D., Dr. Day joined the laboratory of Dr. Chad Mirkin at Northwestern University, where she developed RNA-nanoparticle conjugates known as spherical nucleic acids for gene regulation of glioblastoma. Dr. Day was awarded an International Institute for Nanotechnology Postdoctoral Fellowship and a National Institutes of Health F32 Ruth L. Kirschstein National Research Service Award during her time at Northwestern University. Dr. Day started her lab at the University of Delaware in 2013, and her group investigates the interactions between nanoparticles and biological systems to create novel engineering tools for high precision cancer therapy. She has received numerous grants to support her work, including a W.M. Keck Foundation Grant, an NIH/NIGMS R35 Outstanding Investigator Award, and an NSF CAREER.

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This article is part of the 2018 CMBE Young Innovators special issue.

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Goyal, R., Kapadia, C.H., Melamed, J.R. et al. Layer-by-Layer Assembled Gold Nanoshells for the Intracellular Delivery of miR-34a. Cel. Mol. Bioeng. 11, 383–396 (2018). https://doi.org/10.1007/s12195-018-0535-x

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