Immunostimulatory nucleic acid nanoparticles (NANPs) augment protective osteoblast and osteoclast type I interferon responses to Staphylococcus aureus.

Recalcitrant staphylococcal osteomyelitis may be due, in part, to the ability of Staphylococcus aureus to invade bone cells. However, osteoclasts and osteoblasts are now recognized to shape host responses to bacterial infection and we have recently described their ability to produce IFN-ß following S. aureus infection and limit intracellular bacterial survival/propagation. Here, we have investigated the ability of novel, rationally designed, nucleic acid nanoparticles (NANPs) to induce the production of immune mediators, including IFN-ß, following introduction into bone cells. We demonstrate the successful delivery of representative NANPs into osteoblasts and osteoclasts via endosomal trafficking when complexed with lipid-based carriers. Their delivery was found to differentially induce immune responses according to their composition and architecture via discrete cytosolic pattern recognition receptors. Finally, the utility of this nanoparticle technology was supported by the demonstration that immunostimulatory NANPs augment IFN-ß production by S. aureus infected bone cells and reduce intracellular bacterial burden.

Expanding Structural Space for Immunomodulatory Nucleic Acid Nanoparticles (Nanps) via Spatial Arrangement of Their Therapeutic Moieties.

Different therapeutic nucleic acids (TNAs) can be unified in a single structure by their elongation with short oligonucleotides designed to self-assemble into nucleic acid nanoparticles (NANPs). With this approach, therapeutic cocktails with precisely controlled composition and stoichiometry of active ingredients can be delivered to the same diseased cells for enhancing pharmaceutical action. In this work, an additional nanotechnology-based therapeutic option that enlists a biocompatible NANP-encoded platform for their controlled patient-specific immunorecognition is explored. For this, a set of representative functional NANPs is extensively characterized in vitro, ex vivo, and in vivo and then further analyzed for immunostimulation of human peripheral blood mononuclear cells freshly collected from healthy donor volunteers. The results of the study present the advancement of the current TNA approach toward personalized medicine and offer a new strategy to potentially address top public health challenges related to drug overdose and safety through the biodegradable nature of the functional platform with immunostimulatory regulation.

Induction of Cytokines by Nucleic Acid Nanoparticles (NANPs) Depends on the Type of Delivery Carrier.

Recent insights into the immunostimulatory properties of nucleic acid nanoparticles (NANPs) have demonstrated that variations in the shape, size, and composition lead to distinct patterns in their immunostimulatory properties. While most of these studies have used a single lipid-based carrier to allow for NANPs' intracellular delivery, it is now apparent that the platform for delivery, which has historically been a hurdle for therapeutic nucleic acids, is an additional means to tailoring NANP immunorecognition. Here, the use of dendrimers for the delivery of NANPs is compared to the lipid-based platform and the differences in resulting cytokine induction are presented.

Cracking the Code: Enhancing Molecular Tools for Progress in Nanobiotechnology.

Nature continually refines its processes for optimal efficiency, especially within biological systems. This article explores the collaborative efforts of researchers worldwide, aiming to mimic nature's efficiency by developing smarter and more effective nanoscale technologies and biomaterials. Recent advancements highlight progress and prospects in leveraging engineered nucleic acids and proteins for specific tasks, drawing inspiration from natural functions. The focus is developing improved methods for characterizing, understanding, and reprogramming these materials to perform user-defined functions, including personalized therapeutics, targeted drug delivery approaches, engineered scaffolds, and reconfigurable nanodevices. Contributions from academia, government agencies, biotech, and medical settings offer diverse perspectives, promising a comprehensive approach to broad nanobiotechnology objectives. Encompassing topics from mRNA vaccine design to programmable protein-based nanocomputing agents, this work provides insightful perspectives on the trajectory of nanobiotechnology toward a future of enhanced biomimicry and technological innovation.

Characterization of RNA Nanoparticles and Their Dynamic Properties Using Atomic Force Microscopy.

The protocol described in this chapter allows for acquiring topography images of RNA-based nanoring structures and assessing their dynamic properties using atomic force microscopy (AFM) imaging. AFM is an indispensable tool for characterization of nucleic acid-based nanostructures with the exceptional capability of observing complexes in the range of a few nanometers. This method can visualize structural characteristics and evaluate differences between individual structurally different RNA nanorings. Due to the highly resolved AFM topography images, we introduce an approach that allows to distinguish the differences in the dynamic behavior of RNA nanoparticles not amenable to other experimental techniques. This protocol describes in detail the preparation procedures of RNA nanostructures, AFM imaging, and data analysis.

Characterization of PAMAM Dendrimers for the Delivery of Nucleic Acid Nanoparticles.

The protocols described in this book chapter are meant to be used as an outline and guideline to explore the use of a cationic, polymeric, and synthetic carrier-poly (amidoamine) (PAMAM) dendrimers. The amine-terminated, hyperbranched structures have been identified as a vehicle for the delivery of nucleic acids. As such, clear protocols for the optimization of dendrimer usage should be set in place. This chapter details the experiments used to determine the ratio that dendrimers and nucleic acids should be complexed at through the use of binding assays, nuclease protection assays, and competitive binding assays.

Nanoscale metal-organic frameworks for the delivery of nucleic acids to cancer cells.

Therapeutic nucleic acids (TNAs) are gaining increasing interest in the treatment of severe diseases including viral infections, inherited disorders, and cancers. However, the efficacy of intracellularly functioning TNAs is also reliant upon their delivery into the cellular environment, as unmodified nucleic acids are unable to cross the cell membrane mainly due to charge repulsion. Here we show that TNAs can be effectively delivered into the cellular environment using engineered nanoscale metal-organic frameworks (nanoMOFs), with the additional ability to tailor which cells receive the therapeutic cargo determined by the functional moieties grafted onto the nanoMOF's surface. This study paves the way to integrate the highly ordered programmable nucleic acids into larger-scale functionalized nanoassemblies.