Spherical nucleic acids (SNAs) are highly oriented, well organized, polyvalent structures of nucleic acids conjugated to hollow or solid core nanoparticles. Because they can transfect many tissue and cell types without toxicity, induce minimum immune response, and penetrate various biological barriers (such as the skin, blood-brain barrier, and blood-tumor barrier), they have become versatile tools for the delivery of nucleic acids, drugs, and proteins for various therapeutic purposes. This article describes the unique structures and properties of SNAs and discusses how these properties enable their application in gene regulation, immunomodulation, and drug and protein delivery. It also summarizes current efforts towards clinical translation of SNAs and provides an expert opinion on remaining challenges to be addressed in the path forward to the clinic.

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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.

Educating our immune system via vaccination is an attractive approach to combat infectious diseases. Eliciting antigen specific cytotoxic T cells (CTLs), CD8+ effector T cells, is essential in controlling intracellular infectious diseases such as influenza (Flu), tuberculosis (TB), hepatitis, and HIV/AIDS, as well as tumors. However, vaccination utilizing subunit peptides to elicit a potent CD8+ T cell response with antigenic peptides is typically ineffective due to poor immunogenicity. Here we have engineered a reduction sensitive nanoparticle (NP) based subunit vaccine for intracellular delivery of an antigenic peptide and immunostimulatory adjuvant. We have co-conjugated an antigenic peptide (ovalbumin-derived CTL epitope [OVA257–264: SIINFEKL]) and an immunostimulatory adjuvant (CpG ODNs, TLR9 agonist) to PEG hydrogel NPs via a reduction sensitive linker. Bone-marrow derived dendritic cells (BMDCs) treated with the SIINFEKL conjugated NPs efficiently cross-presented the antigenic peptide via MHC-I surface receptor and induced proliferation of OT-I T cells. CpG ODN-conjugated NPs induced maturation of BMDCs as evidenced by the overexpression of CD80 and CD40 costimulatory receptors. Moreover, codelivery of NP conjugated SIINFEKL and CpG ODN significantly increased the frequency of IFN-γ producing CD8+ effector T cells in mice (~6-fold improvement over soluble antigen and adjuvant). Furthermore, the NP subunit vaccine-induced effector T cells were able to kill up to 90% of the adoptively transferred antigenic peptide-loaded target cell. These results demonstrate that the reduction sensitive NP subunit vaccine elicits a potent CTL response and provide compelling evidence that this approach could be utilized to engineer particulate vaccines to deliver tumor or pathogen associated antigenic peptides to harness the immune system to fight against cancer and infectious diseases.