Kirill A. Afonin

Intracellular bacterial infections, such as those caused by S. aureus, are particularly challenging to treat because pathogens reside within host cells, evading immune defenses and limiting antibiotic efficacy, subsequently promoting drug resistance. To address this, we developed immunostimulatory nucleic acid nanoparticles (NANPs) functionalized with antibacterial silver nanoclusters to enable a dual mechanism of antibacterial action. These NANPs are engineered to activate RIG-I-mediated innate immune responses, which are often bypassed by intracellular bacteria to avoid host clearance. The embedded DNA-templated and -stabilized fluorescent silver nanoclusters, composed of approximately ten silver atoms, exhibit intrinsic antibacterial activity. We systematically evaluated different architectural configurations of silver nanoclusters within NANPs and assessed their relative stabilities, immunostimulatory potentials, and effects against planktonic bacteria and biofilms. We also confirmed that enhanced host immune activation and direct antibacterial activity result in a significant reduction of intracellular S. aureus in primary murine osteoblasts, highlighting the therapeutic potential of this platform.

Introduction: Bacterial meningitis is a rapidly progressing and often fatal infection of the central nervous system (CNS), driven by glial cell activation and potent neuroinflammatory responses. While many pattern recognition receptors are well characterized in CNS immunity, the role of cytosolic nucleic acid sensors such as retinoic acid-inducible gene I (RIG-I) remains largely undefined. Although classically associated with antiviral defense, emerging evidence indicates that RIG-I can also detect bacterial nucleic acids. Methods: To investigate the role of RIG-I during bacterial meningitis, primary murine microglia, astrocytes, and human microglia were infected with Neisseria meningitidis or Streptococcus pneumoniae in the absence or presence of RIG-I-targeting siRNA, recombinant IFN-β, or RIG-I agonists. Whole-cell lysates and supernatants were analyzed for RIG-I and interferon-stimulated gene (ISG) expression by Western blot, cytokine levels (IL-6 and IFN-β) by ELISA and bacterial burden by plating viable CFU. Results: We demonstrate that RIG-I is constitutively expressed in human and murine glial cells and is upregulated upon bacterial infection, with protein levels varying by pathogen. RIG-I activation enhanced IFN production and ISG induction, leading to reduced bacterial burden in an IFNAR-dependent manner. Treatment with RIG-I agonists further amplified these protective responses. Additionally, DNA-templated silver nanoclusters (DNA-AgNCs) served as a complementary antimicrobial approach, exhibiting potent bactericidal activity against N. meningitidis and S. pneumoniae in infected glia. Conclusion: Together, these findings identify RIG-I as a key cytosolic sensor driving protective IFN-dependent antimicrobial responses in glial cells, while AgNCs offer a direct antimicrobial strategy. Collectively, these approaches highlight the potential for targeting both innate immune pathways and direct antimicrobial mechanisms to enhance pathogen control during bacterial meningitis. Funding Source: R03 NS120047-01A1 Topic Categories: Neuroimmunology (NEUR)

The convergence of chemistry, molecular biology, nanotechnology, and computational sciences has created unique opportunities for transformative therapeutics and biomedical devices that are rapidly ...

We introduce RNA‐DNA fusomers, a new class of chemically synthesized oligonucleotides that combine the versatile properties of RNA and DNA within a single sequence and self‐assemble into higher‐order functional structures via a simple one‐pot annealing reaction. This hybrid platform allows precise customization with therapeutic nucleic acids, offering tunable physicochemical, mechanical, and immunological properties, cost‐effective production, and the capacity to integrate biological functionalities intrinsic to both RNA and DNA. The modular architecture of fusomers enables straightforward optimization for diverse biomedical applications, including gene silencing, anti‐inflammatory therapy, anticoagulation, antibacterial activity, and protein biosensing. We demonstrate efficient delivery and intracellular modulation by fusomers in multiple model systems, including human peripheral blood mononuclear cells isolated from healthy human donors and 3D organ‐on‐a‐chip models. Molecular dynamics simulations further elucidate the structural behavior of fusomers and their intended interactions with protein targets. Collectively, these findings position fusomers as a next‐generation therapeutic platform with broad transformative potential.

Establishing a rapid, broad-spectrum antiviral state in human cells offers a promising strategy to combat viral infections, especially when vaccines or pathogen-specific treatments are unavailable. Here, we evaluate immunostimulatory nucleic acid nanoparticles (iNANPs), identified as potent innate immune activators, for their ability to induce protective antiviral states. By mimicking pathogen-associated molecular patterns, iNANPs engage intracellular pattern recognition receptors to stimulate type I and III interferon responses. We tested iNANPs for antiviral efficacy against a replication-incompetent lentiviral vector pseudotyped with vesicular stomatitis virus (VSV) G protein, as well as replication-competent viruses, including VSV, Sendai virus (SeV), and respiratory syncytial virus (RSV). These viruses vary in their mechanisms of innate immune activation and evasion, providing a robust system to assess iNANP activity. Our results demonstrate that iNANPs dramatically restrict viral infection via induction of a robust IFN response, establishing an antiviral state that impairs replication of all tested viruses. This study highlights the potential of iNANPs as a broad-spectrum antiviral prophylactic platform. IMPORTANCE: Establishing a rapid, broad-spectrum antiviral state in human cells offers a promising strategy to combat viral infections, especially when vaccines or pathogen-specific treatments are unavailable. Here, we evaluate immunostimulatory nucleic acid nanoparticles (iNANPs) identified as potent innate immune activators for their ability to induce protective antiviral states. The highly modular design and tunable physicochemical properties of iNANPs with defined architectures and compositions can be tailored to engage specific innate immune sensors. This same modularity allows a seamless “plug-and-play” integration of diverse therapeutic nucleic acids, as well as other therapeutics and small molecules, directly into the iNANP structure. We tested iNANPs for antiviral efficacy against a replication-incompetent lentiviral vector pseudotyped with vesicular stomatitis virus (VSV) G protein, as well as replication-competent viruses, including VSV, Sendai virus (SeV), and respiratory syncytial virus (RSV). Our results demonstrate that iNANPs significantly restrict viral infection, highlighting their potential as a broad-spectrum antiviral prophylactic platform.

Antibiotic-resistant bacteria cause more than one million deaths annually worldwide. The rapid evolution and horizontal gene transfer among pathogens frequently render newly developed antibiotics ineffective shortly after their introduction, underscoring the urgent need for alternative therapeutic strategies. Nanoscale silver is well known for its innate antimicrobial activity but typically requires high concentrations for efficacy that causes toxicities and limits broader clinical applications. To overcome these limitations, we introduce programmable, self-assembling DNA scaffolds that template, stabilize, and spatially organize multiple copies of monodisperse silver nanoclusters (DNA-AgNCs). These nanoscale assemblies enhance the antimicrobial potency of formulations while exhibiting intrinsic fluorescence, providing a dual functionality for therapeutic and fluorescence probing applications. Comprehensive characterization revealed DNA-AgNCs with superior stability and potent activity against clinically relevant antibiotic-resistant ESKAPE pathogens. Also, DNA-AgNCs significantly reduced the intracellular bacterial burden in primary murine bone cells infected with Staphylococcus aureus. Mechanistic studies indicate that bacterial killing by DNA-AgNCs is mediated by reactive oxygen species, particularly singlet oxygen, in conjunction with the disruption of the bacterial membrane. Furthermore, DNA-AgNCs retained strong antibacterial activity after 4 weeks of storage at ambient temperatures, with minimal loss of efficacy. Collectively, these findings establish spatially organized DNA-AgNCs as a promising, modular platform for next-generation antibacterials with integrated real-time fluorescence probing capabilities.

The therapeutic potential of RNAi is limited by the lack of safe, scalable, and efficient delivery systems. Although extracellular vesicles (EVs) are promising carriers for RNA therapeutics, poor scalability and low cargo-loading efficiency constrain their clinical translation. Here, we introduce fully synthetic EV-mimetic (EVM) carriers for the delivery of RNAi-inducing RNA nanoparticles to tumor cells. We systematically evaluated four different EVM cargo-loading strategies using representative RNA nanoparticles and appropriate controls. For each strategy, EVM size, concentration, and cargo encapsulation efficiency were assessed. Cellular uptake and RNA delivery were examined in melanoma 3D spheroids and compared with natural tumor-derived EVs, with EVMs demonstrating superior delivery efficiency. Functional RNAi activation was evaluated in tumor monolayers and 3D spheroids, where EVMs exhibited effective gene silencing with lower toxicity than conventional lipid-based carriers. Overall, our findings establish EVMs as safe and scalable biomimetic platforms for therapeutic nucleic acid delivery and identify optimal loading strategies and RNA nanoparticle designs for achieving maximal gene-silencing.

Antimicrobial resistance is growing among the causative agents of bacterial meningitis, Neisseria meningitidis and Streptococcus pneumoniae, which trigger detrimental neuroinflammatory responses within the central nervous system. Here, we evaluated the antimicrobial potential of DNA-templated and stabilized silver nanoclusters (AgNCs). AgNCs were templated on a single hairpin (HP) or fibrous hairpin structures (HP-F). HP-F provided higher local concentrations of AgNCs, when compared to HP, and exhibited stable physicochemical properties and potent antimicrobial activity. Furthermore, at low silver concentrations, AgNCs restricted bacterial survival and reduced inflammatory responses in microglia without causing cytotoxicity, supporting further development of AgNCs as therapeutics for meningitis.

The rise of personalized medicine demands affordable biomaterials with tunable physicochemical properties, biological activities, and immune responses. We introduce a nucleic acid nanoparticle (NANP)‐based platform designed to engage with the human immune system in a controllable manner. By varying the chemical composition while keeping the architectural parameters constant, we identified key factors that influence the immunorecognition and stability of NANPs. Chemical modifications provide additive functional regulation, while pairing NANPs with different delivery agents further enhances their immunomodulatory capabilities. Specific compositions boost T cell effector functions, promote B cell proliferation, and activate innate immune populations in human peripheral blood mononuclear cells. The resulting interferon responses and immune signatures mimic those triggered by viral infections. Notably, in vivo studies demonstrate that NANPs function as efficiently as traditional clinically used adjuvants, without increasing the risk of autoimmunity. This suggests that NANPs are a safe and effective tool for immunotherapy with promise for developing formulations targeting cancer and infectious diseases.

Rapid advances in biomedical research and nanomedicine have driven the development and implementation of New Approach Methodologies (NAMs) for predictive evaluations of novel formulations.