3D-Nanoprinted Glass Microneedle Technologies for Microinjection Applications

Abstract

Hollow glass microneedles are critical tools in biomedical research and clinical applications, enabling the delivery of fluidic payloads into biological targets. The efficacy of current microneedle technologies is limited by conventional microneedle fabrication methods that not only rely on manual processes that introduce undesired tip variability, but more importantly, they produce microneedles with a single opening at the top of the tip, which are prone to clogging. Consequently, there is a need for new fabrication strategies that are capable of reliably manufacturing microneedle geometries that mitigate clogging. Direct Laser Writing (DLW) is a two-photon polymerization-based 3D nanoprinting technique capable of producing features down to 100 nm, and is thus uniquely suited for fabricating microneedles with sophisticated architectures capable of preventing clogging phenomena. However, conventional polymer photoresins lack the mechanical properties required for penetrating a number of critical biological targets, such as the chorion membrane of zebrafish embryos. Hybrid organic-inorganic photopolymers, particularly those based on Polyhedral Oligomeric Silsesquioxane (POSS), offer a potential solution but remain underexplored for biomedical microneedle applications. This dissertation establishes a new class of 3D-nanoprinted hollow glass microneedles suitable for zebrafish embryo microinjections through three contributions. First, we identify a previously unreported geometric size constraint for POSS-based glass structures, wherein features thicker than 30 µm crack during thermal processing, compromising optical transparency and mechanical integrity. Then, we leveraged this newfound knowledge to develop design and fabrication strategies for POSS-based glass microneedle technologies for targets inaccessible to standard photoresins. Lastly, we realized anti-clogging POSS-based hollow glass microneedles and validated their functionality in vivo through microinjection of CRISPR-Cas9 into zebrafish embryos, demonstrating successful penetration, delivery, and withdrawal, with subsequent phenotypic evidence of successful mutagenesis. These results suggest that DLW-based 3D-nanoprinted POSS-based glass microneedles hold unique potential as a robust platform for high-performance microinjection applications, particularly for those into hard-to-puncture biological targets.