Natalia Chernyak

Immunomodulatory nucleic acids have extraordinary promise for treating disease, yet clinical progress has been limited by a lack of tools to safely increase activity in patients. Immunomodulatory nucleic acids act by agonizing or antagonizing endosomal toll-like receptors (TLR3, TLR7/8, and TLR9), proteins involved in innate immune signaling. Immunomodulatory spherical nucleic acids (SNAs) that stimulate (immunostimulatory, IS-SNA) or regulate (immunoregulatory, IR-SNA) immunity by engaging TLRs have been designed, synthesized, and characterized. Compared with free oligonucleotides, IS-SNAs exhibit up to 80-fold increases in potency, 700-fold higher antibody titers, 400-fold higher cellular responses to a model antigen, and improved treatment of mice with lymphomas. IR-SNAs exhibit up to eightfold increases in potency and 30% greater reduction in fibrosis score in mice with nonalcoholic steatohepatitis (NASH). Given the clinical potential of SNAs due to their potency, defined chemical nature, and good tolerability, SNAs are attractive new modalities for developing immunotherapies.

A novel class of metal-free spherical nucleic acid nanostructures was synthesized from readily available starting components. These particles consist of 30 nm liposomal cores, composed of an FDA-approved 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid monomer. The surface of the liposomes was functionalized with DNA strands modified with a tocopherol tail that intercalates into the phospholipid layer of the liposomal core via hydrophobic interactions. The spherical nucleic acid architecture not only stabilizes these constructs but also facilitates cellular internalization and gene regulation in SKOV-3 cells.

Alloy nanoparticles are important in many fields, including catalysis, plasmonics, and electronics, due to the chemical and physical properties that arise from the interactions between their components. Typically, alloy nanoparticles are made by solution-based synthesis; however, scanning-probe-based methods offer the ability to make and position such structures on surfaces with nanometer-scale resolution. In particular, scanning probe block copolymer lithography (SPBCL), which combines elements of block copolymer lithography with scanning probe techniques, allows one to synthesize nanoparticles with control over particle diameter in the 2-50 nm range. Thus far, single-element structures have been studied in detail, but, in principle, one could make a wide variety of multicomponent systems by controlling the composition of the polymer ink, polymer feature size, and metal precursor concentrations. Indeed, it is possible to use this approach to synthesize alloy nanoparticles comprised of combinations of Au, Ag, Pd, Ni, Co, and Pt. Here, such structures have been made with diameters deliberately tailored in the 10-20 nm range and characterized by STEM and EDS for structural and elemental composition. The catalytic activity of one class of AuPd alloy nanoparticles made via this method was evaluated with respect to the reduction of 4-nitrophenol with NaBH4. In addition to being the first catalytic studies of particles made by SPBCL, these proof-of-concept experiments demonstrate the potential for SPBCL as a new method for studying the fundamental science and potential applications of alloy nanoparticles in areas such as heterogeneous catalysis.

Abstract Immunostimulatory spherical nucleic acids (IS‐LSNAs) comprised of RNA selective for toll‐like receptors (TLRs) 7/8 are synthesized and characterized. These structures consist of liposomal cores functionalized with a dense shell of RNA inserted into the wall of the lipid core via hydrophobic cholesterol moieties. IS‐LSNAs potently activate TLR7/8 via NF‐κΒ signaling in reporter cell lines and in primary immune cells as evidenced by cytokine production and the upregulation of costimulatory receptors. Importantly, they are preferentially taken up by plasmacytoid dendritic cells, an observation that makes them potentially useful for immunotherapy. In addition, these structures contain a core that can be loaded with antigens and used to prime T cells. In this regard, it is shown that dendritic cells treated with IS‐SNAs loaded with ovalbumin peptide can prime ova specific CD8 + T cells. In addition to introducing the first IS‐LSNAs consisting of RNA, these experiments show that one can facilitate an antigen‐specific T cell response greater than that of free or cationic lipid‐transfected RNA of the same sequence selective for TLR7/8. This work points toward the promise of using IS‐LSNAs comprised of RNA as potent and highly tunable TLR‐specific agents for the development of vaccines and other pharmaceuticals that require selective immunomodulation.

Cysteines are routinely used as site-specific handles to synthesize antibody–drug conjugates for targeted immunotherapy applications. Michael additions between thiols and maleimides are some of the most common methods for modifying cysteines, but these functional groups can be difficult to prepare on scale, and the resulting linkages have been shown to be reversible under some physiological conditions. Here, we show that the enzyme tyrosinase, which oxidizes conveniently accessed phenols to afford reactive ortho-quinone intermediates, can be used to attach phenolic cargo to cysteines engineered on antibody surfaces. The resulting linkages between the thiols and ortho-quinones are shown to be more resistant than maleimides to reversion under physiological conditions. Using this approach, we construct antibody conjugates bearing cytotoxic payloads, which exhibit targeted cell killing, and further demonstrate this method for the attachment of a variety of cargo to antibodies, including fluorophores and oligonucleotides.