A Fluorescence Spectroscopic Approach to Studying Nanoparticle Chemical Environments, Architecture, and Assembly

Abstract

Three decades of intense research in nanomedicine have yielded few clinical outcomes, particularly in targeted gene delivery. Beyond regulatory challenges, the scarcity of FDA-approved nanoformulated gene therapies (only three as of writing) can be attributed to several technical issues: 1) the complex, multidimensional chemical and materials design space of nanoparticle delivery systems makes it difficult to determine the appropriate nanoparticle physicochemical characteristics for therapeutic applications, 2) successful therapeutic payload delivery to target sites requires nanoparticles to navigate complex biological environments and barriers, and 3) current analytical tools provide limited insight into nanoparticle interactions with these biological environments. Thus, new methods are needed to study nanoparticle-environment interactions to better inform nanomedicine design. This dissertation focuses on time-resolved fluorescence spectroscopy (TRFS) to study therapeutically relevant nanoparticles, including their formulation, interactions, architecture, and assembly. In Chapter 2, a photoreactive electrohydrodynamic (EHD) jetting process was developed to prepare synthetic protein nanoparticles, achieving a four-order magnitude improvement in processing and purification time. This process utilized a fluorescent, small molecule photocrosslinker with two photoreactive benzophenone-containing arms extending from a dithiomaleimide core. The photocrosslinker stabilized protein nanoparticles and served as an intrinsic molecular reporter for formulation monitoring using TRFS, providing insights into nanoparticle stability and architecture inaccessible to state-of-the-art techniques like scanning electron microscopy and dynamic light scattering. In Chapter 3, the fluorescent, small molecule photocrosslinker’s design was refined to enhance reactivity, as two variants were developed for preparing nanoparticles from both protein and non-protein materials, specifically synthetic polymers. One crosslinker contained benzophenone moieties with four reactive arms based on the same dithiomaleimide core as in Chapter 2, while the second variant contained methacrylate reactive groups. Three distinct nanoparticle types – protein nanoparticles, polymer nanogels, and block copolymer micelles – were prepared using the refined crosslinkers. Each nanoparticle type could discern minute changes in interparticle chemical environments, as measured by TRFS, with protein nanoparticles exhibiting the greatest sensitivity across an order of magnitude. Encapsulation of the small molecule drug paclitaxel in protein nanoparticles induced characteristic changes in fluorescence lifetime profiles based on the drug encapsulation mode. Finally, a heterobifunctional reversible addition–fragmentation chain-transfer (RAFT) system was developed for the controlled polymerization of methacrylic monomers, enabling orthogonal end group conjugations post-polymerization. This versatile system was used to synthesize a variety of linear, monodisperse polymers with different chemical characters, confirming functionality through the conjugation of two different small molecule fluorescent probes to the functional end groups. An amphiphilic block copolymer was synthesized, fluorescent end group conjugated, and self-assembled into micelles. TRFS was used to probe micelle assembly by measuring the fluorescence lifetime of both end groups simultaneously, as well as the micelle system’s interaction with 5 nm silver nanoparticles. Self-reporting nanoparticles, such as the ones developed in this dissertation, will be critical for unraveling nanoparticle stability and nanoparticle-drug interactions informing the future development of rationally engineered nanoparticle-based drug carriers.