Electrohydrodynamic Jetted Biomaterials with Cell/Tissue Specificity for Gene Therapy and Regenerative Medicine

Abstract

Electrohydrodynamic (EHD) jetting is a promising technique developed from the conventional electrospinning. Recent advancements in EHD jetting have enabled the production of uniform particles in the nano-scale regime, as well as precise, micron- or submicron-scale fiber deposition in a layer-by-layer fashion. Over the last decade we have witnessed the exponential growth of non-viral gene therapies, in particular utilizing nanoparticles for the COVID-19 mRNA vaccines, cancer therapies, and in the treatment of rare genetic disorders. The first bottleneck in the development of non-viral vectors is the packaging of long anionic nucleic acid chains into a small particle, i.e., plasmid DNA (pDNA) complexation and condensation. The second crucial step for success is the functional delivery of therapeutic nuclei acid into cells. In Chapter 2, EHD jetting is first leveraged for particle formation via rapid solidification and condensation of solution, and then particle surface capping, using low levels of cationic polymers, to stabilize and suspend the final surface-capped protein nanoparticles (scPNPs). The role of gene vector loading and particle dosage on pDNA transfection performance for this scPNP system was then assessed, with an aim to optimize design principles for improving the efficiency and biosafety of the scPNPs. Furthermore, this included the development of a high-throughput flow cytometry-based workflow for accurate quantification of the cellular uptake. This methodological approach permitted attainment of the uptake kinetic profiles for scPNPs, revealing a highly efficient internalization process. This work also highlights the importance of reporting NP number as a main descriptor for dosage. Finally, using pharmacological inhibitors, this study identified which endocytosis pathways contribute most to the enhanced transfection and cellular uptake of scPNPs, highlighting similarities and differences between cells in vitro and in vivo. Overall, this work aims to employ crosslinker-free scPNP systems to achieve a wide range of pDNA loading levels and, together with particle number, pursue optimized parameters to minimize cytotoxicity and maximize transfection efficiency. Moreover, this work advances the fundamental understanding of design choices when optimizing for the delivery of large molecular weight nucleic acids to different type of cells/tissues. Nearly half of individuals in the US aged 30 and older have periodontitis, resulting in the destruction of supporting periodontal tissues around the teeth, including both soft tissue and hard tissue, as well as the interface in between. In Chapter 3, by leveraging the power of EHD jetting to fabricate highly porous, highly ordered and hierarchical scaffolds, multicompartmental structures were proven to coordinate the proliferation and differentiation of multiple cell types. It is the first reported attempt to employ melt electrowriting (MEW), a specific subtype of EHD jetting of melts, to fabricate multicompartmental scaffolds comprising of bone compartment, periodontal ligament (PDL) compartment, and transitional region for periodontal regeneration. Careful consideration and design of geometric factors promoted cell proliferation, cell expression, cell alignment, and extracellular biomolecule alignment in vitro all in keeping with early-stage periodontal regeneration in vivo. The 3D structure-induced cell behaviors promoted a gradient transition from calcified to uncalcified regions with longer term growth, effectively recapitulating the key features of native interfacial tissues in periodontium. This multicompartmental approach reveals the importance of developing 3D tissue-engineered constructs that better mimic the physical structure of native tissues and advances the development of next-generation scaffolds for interfacial and multi-tissue engineering.