Engineering Sub-20 nm Nanocarriers (3-helix Micelles) from the Molecular to Systemic Physiological Levels

Cancer nanomedicine was developed with the promise to deliver therapeutic cargo safely to diseased tissue. In over forty years, an impressive smattering of creative designs has emerged for particles of varying materials, surface properties, and targeting strategies, yet only few successfully made it through clinical trials. These failures can be attributed to flaws in the fundamental science, clinical trial designs, and market competition. Thusly, we sought to fill knowledge gaps in the areas of particle design, in vivo circulation, and delivery into the tumor parenchyma. We accomplished this via in-depth examination of nanocarrier internal structure, modeling nanoparticle tumor transport, and exploring fractal tumor vascular ultrastructures./nNanocarrier development ought to be informed by biological constraints. A greater appreciation for size exclusion limitations in tumor extravasation and penetration has led to small nanocarriers (10 - 30 nm) being favored recently. This size range can still minimize kidney glomerular filtration and reticuloendothelial system clearance. However, such small nanocarrier dimensions begin to approach that of the small molecules they are intended to carry. As such, the cargo can have a large contribution to the overall particle’s properties. Using sub-20 nm three-helix micelles (3HM) as model particles, we examined the interplay of properties arising from cargo-carrier co-assembly. The results revealed that both hydrophobicity and geometry dictate compatibility between small molecules and 3HM’s alkyl tail core. This proximally fitted assembly creates a stable architecture with increased alkyl tail crystallinity. With ~50x greater cargo preference for the micelle core than the bulk solvent, a slow release profile is achieved, advantageous for sequestering the therapeutic payload prior to release in target tissues./nAt the systemic biodistribution level, factors impacting nanoparticle distribution in the tumor must be examined. While some nanoparticles may circulate for extended periods of time in the blood following intravenous injection, it does not guarantee that they have higher tumor accumulation than other types of particles. Among other unique microenvironment properties, tumors feature leaky vessels. This enables nanoparticles to extravasate in a passively-targeted fashion, leading to the enhanced permeability and retention (EPR) effect. However, EPR permeability is different for different tumors, and for different particles. It is important to quantify and compare this factor among different candidate nanocarriers, in order to optimize the best design. Thus far, permeability has been difficult to measure in situ, and existing mathematical models of particle tumor transport are often too complicated and inaccessible. As such, we have developed a simpler mathematical model called diffusive flux modelling (DFM), applicable to routine biodistribution data available for most published nanoparticles. DFM decouples contributions of plasma pharmacokinetics and tumor vascular permeability to explain the observed particle tumor accumulation. Using this method, we have made a semi-quantitative comparison across various tumor models, particle size, and active targeting strategies. To maximize accessibility, the DFM analysis code is made available online with instruction manuals at https://github.com/mlim0789/DFM-Fitting./nIn-depth ultrastructural examination of the tumor vasculature is necessary to understand differences in nanoparticle permeabilities. Taking the worst-case scenario of a poorly-permeable tumor with limited treatment options, we focused our efforts on glioblastoma (GBM) brain tumors. Electron microscopy studies were conducted with a clinically-relevant patient-derived xenograft model, GBM6FL, inoculated intracranially in mice. The results demonstrated that both inter- and intra-tumor vascular heterogeneity impacted nanoparticle tumor penetration and treatment upon dosing of 3HM-encapsulated Doxorubicin. Three classes of GBM vessels were identified, with decreasing junction integrity, and increasing membrane curvature and fractal dimensions. Narrow endothelial junction gaps and intercellular spacing in the tumor parenchyma impedes the migration of particles >20 nm, which explains the ineffectiveness of 100 nm liposomal therapy. The fractal nature of vessel morphology at the macro and nano-scale suggests that angiography studies for vascular tortuosity can be used to predict the permeability of passively-targeted nanoparticle therapy. This can be valuable for patient stratification during clinical studies, and selection of applicable nanoparticle therapies, especially when coupled with genetic profiling./nBuilding upon the discoveries presented in this work, we proposed future studies that will shed light on nanocarrier and tumor dynamics to optimize treatment strategies. This involves additional ultrastructural examination of tumors at varying stages of growth and tracking the tumor accumulation of particles with varying disassembly kinetics. These new pieces of information will be combined with particle physicochemical characteristics, cellular uptake timescales, tumor doubling time, and vascular permeability modeling to ultimately assemble a comprehensive semi-quantitative model. We envision that this tool will be useful for designing the ideal particle and dosing strategy that maximizes efficacy./nIn conclusion, while cancer nanomedicine development has been met with numerous roadblocks, opportunities still abound to realize its true potential. Insights into particle design, in vivo circulation, and tumor structures that are presented here goes towards a global treatment strategy informed by materials science and tumor biology. Coupled with advances from the broader oncological, immunological, and biophysical research community, we hope to achieve reliable and successful treatment outcomes for cancer patients.

Advisor: Xu, Ting