Development and Validation of Scalable Nanoplatforms for Anti-Inflammatory Immunotherapy

Allen, Sean David

doi:https://doi.org/10.21985/n2-dfbd-a538

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Development and Validation of Scalable Nanoplatforms for Anti-Inflammatory Immunotherapy

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Nanocarriers are nanometer-sized (1-1000 nm) structures capable of encapsulating cargo. This encapsulation can drastically alter the pharmacokinetic properties of the cargo, while also allowing for the rational design and engineering of the nanocarrier itself. Poly(ethylene glycol)-block-poly(propylene sulfide) is an amphiphilic diblock copolymer capable of self-assembling into diverse nanocarriers. The purpose of this work was to investigate the utility of PEG-b-PPS nanocarriers for anti-inflammatory usage as therapeutic nanocarriers, particularly with regard to the sterile scale-up of their production and their efficacy in ameliorating the inflammatory conditions in atherosclerosis. This work covers the production of PEG-b-PPS nanocarriers, encapsulation of compounds within said nanocarriers, their safety and biodistribution in mice and non-human primates, and their utility as anti-inflammatory therapeutic nanocarriers. To assess these characteristics of the nanocarriers, several quantitative methodologies were utilized. Size and morphological nanocarrier characterization was performed, along with in vitro examination of immunogenicity, compound encapsulation efficiency, delivery and subcellular localization, and assessment of cell viability. Finally, in vivo work, centered firstly around intravital fluorescent imaging and flow cytometric analysis of uptake in organs and cell populations and secondly in assessment of therapeutic efficacy in the ldlr-/- mouse model of atherosclerosis, was performed. I have found that flash nanoprecipitation, a novel method of forming soft polymeric nanostructures, is capable of recapitulating all of the previously formed nanostructures using PEG-b-PPS polymer in addition to a previously unavailable nanostructure called a bicontinuous nanosphere. I have characterized these four different nanostructures and have demonstrated their ability to encapsulate different compounds, particularly the ability of polymersomes and bicontinuous nanospheres to simultaneously encapsulate both hydrophilic and hydrophobic compounds. Flash nanoprecipitation is scalable to production volumes relevant for non-human primate experimentation and is easily performed under sterile conditions. PEG-b-PPS nanocarriers formed by flash nanoprecipitation are non-immunogenic, non-toxic, are taken up primarily by the spleen and liver after IV administration and are taken up primarily by phagocytic cells such as macrophages, monocytes, and dendritic cells. Encapsulation of celastrol, an inhibitor of the inflammatory NF-κB pathway, in these nanocarriers results in the reduction of inflammatory cells in the blood and atherosclerotic plaques of atherosclerotic mice and additionally results in the reduction of plaque staining area. I conclude therefore that PEG-b-PPS nanocarriers are an effective drug delivery platform for the encapsulation and in vivo delivery of anti-inflammatory compounds for therapeutic biomedical applications. Additionally, I have demonstrated that flash nanoprecipitation is a rapid, scalable, and sterile method of forming these nanocarriers for future translational use.

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