RUI: BMAT: Evaluating Ionizable Amphiphilic Janus Dendrimers as Delivery Agents for Nucleic Acids

NON-TECHNICAL SUMMARY: Methods to alter gene expression have the potential to transform modern medicine. These tools directly modify genetic information or change how much or how little a specific gene is expressed. Many of these approaches use molecules called nucleic acids to treat disease, but for these therapies to be successful, small particles or vesicles must encapsulate and carry these nucleic acids to desired cell types. Various biomaterials such as lipids, polymers, and dendrimers have been used to construct these carrier systems. A relevant example of such a system is the COVID-19 vaccine, a lipid-based nanoparticle that delivers messenger RNA (mRNA) to elicit an immune response. Understanding how these biomaterials influence the physical properties of the resulting particles and how they affect interactions within the body is critical to designing effective materials for delivery of nucleic acid therapeutics. This work aims to characterize vesicles made from a new type of molecule called ionizable amphiphilic Janus dendrimers (IAJDs). Janus dendrimers are structures with two different chemical characteristics on each side. The proposed studies will establish how differences in IAJD properties affect vesicle characteristics like size and shape, how these vesicles interact with common biological proteins like those found in the blood stream, and how these vesicles enter and release nucleic acids in cells. More broadly, this project will provide meaningful hands-on research experiences for undergraduate students via summer research opportunities and development of a mini-course based undergraduate research experience (mini-CURE) in biophysical chemistry. Undergraduates will also be encouraged to explore their scientific interests and participate in the broader scientific community via participation in a STEM-focused student club on campus. TECHNICAL SUMMARY: Effective use of nucleic acid therapeutics relies on delivery agents that enhance nucleic acid circulation times, shield from nucleases, and aid in intracellular delivery. In particular, the success of RNA therapeutics, including messenger RNA (mRNA) and small interfering RNA (siRNA), can be attributed to innovations surrounding the ionizable lipid component of the lipid nanoparticle (LNP); however, challenges associated with immunotoxicity, laborious syntheses, and extrahepatic delivery continue to limit the scope of these delivery vehicles, suggesting a need for new ionizable amphiphilic molecules. Recently, ionizable amphiphilic Janus dendrimers (IAJDs) have emerged as tools to encapsulate and deliver mRNA in vivo via a one-component dendrimersome nanoparticle (DNP) delivery system. Initial publications exploring mRNA delivery using DNPs suggest that IAJD structure dictates in vivo localization as well as activity; however, fundamental and mechanistic questions remain. The major goal of this work is to characterize DNPs prepared from IAJDs in the presence and absence of nucleic acid therapeutics (mRNA and siRNA) and understand how these DNPs interact with and within biological systems. Specifically, this work will (1) elucidate how IAJD structure dictates the physical characteristics of DNPs including size, shape, morphology, and nucleic acid encapsulation, (2) determine how these differences influence interactions of DNPs with serum proteins, thus impacting cytotoxicity and immunogenicity, and (3) evaluate mechanisms for cellular internalization and functional delivery of mRNA and siRNA. These studies will establish structure-function relationships to explain how DNPs encapsulating nucleic acids exert their therapeutic effects. This project will provide meaningful hands-on research experiences for undergraduate students via summer research opportunities and development of a mini-course based undergraduate research experience (mini-CURE) in biophysical chemistry. Undergraduates will also be encouraged to explore their scientific interests and participate in the broader scientific community via participation in a STEM-focused student club on campus. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.