Tuning DNA Dissociation from Spherical Nucleic Acids for Enhanced Immunostimulation.

The stability of the core can significantly impact the therapeutic effectiveness of liposome-based drugs. While the spherical nucleic acid (SNA) architecture has elevated liposomal stability to increase therapeutic efficacy, the chemistry used to anchor the DNA to the liposome core is an underexplored design parameter with a potentially widespread biological impact. Herein, we explore the impact of SNA anchoring chemistry on immunotherapeutic function by systematically studying the importance of hydrophobic dodecane anchoring groups in attaching DNA strands to the liposome core. By deliberately modulating the size of the oligomer that defines the anchor, a library of structures has been established. These structures, combined with in vitro and in vivo immune stimulation analyses, elucidate the relationships between and importance of anchoring strength and dissociation of DNA from the SNA shell on its biological properties. Importantly, the most stable dodecane anchor, (C12)9, is superior to the n = 4-8 and 10 structures and quadruples immune stimulation compared to conventional cholesterol-anchored SNAs. When the OVA1 peptide antigen is encapsulated by the (C12)9 SNA and used as a therapeutic vaccine in an E.G7-OVA tumor model, 50% of the mice survived the initial tumor, and all of those survived tumor rechallenge. Importantly, the strong innate immune stimulation does not cause a cytokine storm compared to linear immunostimulatory DNA. Moreover, a (C12)9 SNA that encapsulates a peptide targeting SARS-CoV-2 generates a robust T cell response; T cells raised from SNA treatment kill >40% of target cells pulsed with the same peptide and ca. 45% of target cells expressing the entire spike protein. This work highlights the importance of using anchor chemistry to elevate SNA stability to achieve more potent and safer immunotherapeutics in the context of both cancer and infectious disease.

Multi-antigen spherical nucleic acid cancer vaccines.

Cancer vaccines must activate multiple immune cell types to be effective against aggressive tumours. Here we report the impact of the structural presentation of two antigenic peptides on immune responses at the transcriptomic, cellular and organismal levels. We used spherical nucleic acid (SNA) nanoparticles to investigate how the spatial distribution and placement of two antigen classes affect antigen processing, cytokine production and the induction of memory. Compared with single-antigen SNAs, a single dual-antigen SNA elicited a 30% increase in antigen-specific T cell activation and a two-fold increase in T cell proliferation. Antigen placement within dual-antigen SNAs altered the gene expression of T cells and tumour growth. Specifically, dual-antigen SNAs encapsulating antigens targeting helper T cells and with externally conjugated antigens targeting cytotoxic T cells elevated antitumour genetic pathways, stalling lymphoma tumours in mice. Additionally, when combined with the checkpoint inhibitor anti-programmed-cell-death protein-1 in a mouse model of melanoma, a specific antigen arrangement within dual-antigen SNAs suppressed tumour growth and increased the levels of circulating memory T cells. The structural design of multi-antigen vaccines substantially impacts their efficacy.

Chemically Tuning the Antigen Release Kinetics from Spherical Nucleic Acids Maximizes Immune Stimulation.

Cancer vaccine structure is emerging as an important design factor that offers tunable parameters to enhance the targeted immune response. We report the impact of altering the antigen release rate from spherical nucleic acid (SNA) vaccines-nanoparticles with a liposomal core and surface-anchored adjuvant DNA-on immune stimulation. Peptide antigens were incorporated into SNAs using either a nonreducible linker or one of a series of reduction-triggered traceless linkers that release the native peptide at rates controlled by their substitution pattern. Compared with a nonreducible linkage, the traceless attachment of antigens resulted in lower EC50 of T cell proliferation in vitro and greater dendritic cell (DC) activation and higher T cell killing ability in vivo. Traceless linker fragmentation rates affected the rates of antigen presentation by DCs and were correlated with the in vitro potencies of SNAs. Antigen release was correlated with the ex vivo -log(EC50), and more rapid antigen release resulted in an order of magnitude improvement in the EC50 and earlier and greater antigen presentation over the same time-period. In vivo, increasing the rate of antigen release resulted in higher T cell activation and target killing. These findings provide fundamental insights into and underscore the importance of vaccine structure.

Spherical Nucleic Acid Vaccine Structure Markedly Influences Adaptive Immune Responses of Clinically Utilized Prostate Cancer Targets.

Cancer vaccines, which activate the immune system against a target antigen, are attractive for prostate cancer, where multiple upregulated protein targets are identified. However, many clinical trials implementing peptides targeting these proteins have yielded suboptimal results. Using spherical nucleic acids (SNAs), we explore how precise architectural control of vaccine components can activate a robust antigen-specific immune response in comparison to clinical formulations of the same targets. The SNA vaccines incorporate peptides for human prostate-specific membrane antigen (PSMA) or T-cell receptor γ alternate reading frame protein (TARP) into an optimized architecture, resulting in high rates of immune activation and cytolytic ability in humanized mice and human peripheral blood mononuclear cells (hPBMCs). Specifically, administered SNAs elevate the production and secretion of cytokines and increase polyfunctional cytotoxic T cells and effector memory. Importantly, T cells raised from immunized mice potently kill targets, including clinically relevant cells expressing the whole PSMA protein. Treatment of hPBMCs increases costimulatory markers and cytolytically active T cells. This work demonstrates the importance of vaccine structure and its ability to reformulate and elevate clinical targets. Moreover, it encourages the field to reinvestigate ineffective peptide targets and repackage them into optimally structured vaccines to harness antigen potency and enhance clinical outcomes.

Impact of Liposomal Spherical Nucleic Acid Structure on Immunotherapeutic Function.

Liposomal spherical nucleic acids (L-SNAs) show significant promise as cancer immunotherapeutics. L-SNAs are highly modular nanoscale assemblies defined by a dense, upright radial arrangement of oligonucleotides around a liposomal core. Herein, we establish a set of L-SNA design rules by studying the biological and immunological properties of L-SNAs as a function of liposome composition. To achieve this, we synthesized liposomes where the lipid phosphatidylcholine headgroup was held constant, while the diacyl lipid tail chain length and degree of saturation were varied, using either 1,2-dioleylphosphatidylcholine (DOPC), 1,2-dimyristoyl-phosphatidylcholine (DMPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), or 1,2-distearoyl-phosphatidylcholine (DSPC). These studies show that the identity of the constituent lipid dictates the DNA loading, cellular uptake, serum stability, in vitro immunostimulatory activity, and in vivo lymph node accumulation of the L-SNA. Furthermore, in the 4T1 mouse model of triple-negative breast cancer (TNBC), the subcutaneous administration of immunostimulatory L-SNAs synthesized with DPPC significantly decreases the production of lung metastases and delays tumor growth as compared to L-SNAs synthesized using DOPC, due to the enhanced stability of L-SNAs synthesized with DPPC over those synthesized with DOPC. Moreover, the inclusion of cell lysates derived from Py8119 TNBC cells as antigen sources in L-SNAs leads to a significant increase in antitumor efficacy in the Py8119 model when lysates are encapsulated in the cores of L-SNAs synthesized with DPPC rather than DOPC, presumably due to increased codelivery of adjuvant and antigen to dendritic cells in vivo. This difference is further amplified when using lysates from oxidized Py8119 cells as a more potent antigen source, revealing synergy between the lysate preparation method and liposome composition in synthesizing immunotherapeutic L-SNAs. Together, this work shows that the biological properties and immunomodulatory activity of L-SNAs can be modulated by exchanging liposome components, providing another handle for the rational design of nanoscale immunotherapeutics.

Oriented Attachment Is a Major Control Mechanism To Form Nail-like Mn-Doped ZnO Nanocrystals.

Here, we present a controlled synthesis of Mn-doped ZnO nanoparticles (NPs) with predominantly nail-like shapes, whose formation occurs via tip-to-base-oriented attachment of initially formed nanopyramids, followed by leveling of sharp edges that lead to smooth single-crystalline "nails". This shape is prevalent in noncoordinating solvents such as octadecene and octadecane. Yet, the double bond in the former promotes oriented attachment. By contrast, Mn-doped ZnO NP synthesis in a weakly coordinating solvent, benzyl ether, results in dendritic structures because of random attachment of initial NPs. Mn-doped ZnO NPs possess a hexagonal wurtzite structure, and in the majority of cases, the NP surface is enriched with Mn, indicating a migration of Mn2+ ions to the NP surface during the NP formation. When the NP formation is carried out without the addition of octadecyl alcohol, which serves as a surfactant and a reaction initiator, large, concave pyramid dimers are formed whose attachment takes place via basal planes. UV-vis and photoluminescence spectra of these NPs confirm the utility of controlling the NP shape to tune electro-optical properties.

Enhancing the Catalytic Activity of Zn-Containing Magnetic Oxides in a Methanol Synthesis: Identifying the Key Factors.

A new family of Ni-, Co-, and Cr-doped Zn-containing magnetic oxide nanoparticles (NPs) stabilized by polyphenylquinoxaline (PPQ) and hyperbranched pyridylphenylene polymer (PPP) has been developed. These NPs have been synthesized by thermal decomposition of Zn and doping metal acetylacetonates in the reaction solution of preformed magnetite NPs, resulting in single-crystal NPs with spinel structure. For the PPQ-capped NPs, it was demonstrated that all three types of metal species (Fe, Zn, and a doping metal) reside within the same NPs, the surface of which is enriched with Zn and a doping metal, while the deeper layers are enriched with Fe. The Cr-doped NPs at the high Cr loading are an exception due to favored deposition of Cr on magnetite located in the NP depth. The PPP-capped NPs exhibit similar morphology and crystallinity; however, the detailed study of the NP composition was barred due to the high PPP amount retained on the NP surface. The catalyst testing in syngas conversion to methanol demonstrated outstanding catalytic properties of doped Zn-containing magnetic oxides, whose activities are dependent on the doping metal content and on the stabilizing polymer. The PPP stabilization allows for better access to the catalytic species due to the open and rigid polymer architecture and most likely optimized distribution of doping species. Repeat experiments carried out after magnetic separation of catalysts from the reaction mixture showed excellent catalyst stability even after five consecutive catalytic runs.