WP1. Polymersome Fabrication: Non-immunogenic and biocompatible vesicles called polymersomes were fabricated from synthetic amphiphilic block copolymers (ABCs) to function as artificial organelles. The ABCs were composed of a PEGylated and water-soluble segment conjoined with a water insoluble segment consisting of pH- and photo-sensitive functionalities. The pH sensitivity of the ABC was engineered to promote solubility in water at low pH, and insolubility at higher pH. Therefore, at higher pH the ABC chains undergoes a reversible phase separation and self-assembles into polymersomes. Subjecting the polymersomes to UV irradiation prompts the crosslinking of the photo-sensitive segment, which makes the self-assembly irreversible when subjected to any further pH changes, thus stabilizing the polymersomes. WP2. Biomolecule Release: Polymersomes were designed and fabricated to function as artificial organelles for performing the encapsulation and programmable release of enzymes and proteins under specific physiological conditions. To this end, it was necessary to determine the optimum protein/ enzyme size for sufficient and efficient encapsulation and release processes under various physiological conditions. At optimum protein/ enzyme size, it was necessary to define the permeability of the polymersomes membrane as a function of the membrane's thickness and composition for both deuterated and non-deuterated analogues. Overall, polymersomes as synthetic vesicles are attributed to better mechanical and chemical stability relative to biological alternatives. WP3. Fabrication of Exosome-DNA-Polymer Hybrid: As complimentary alternatives to artificial organelles, biologically produced vesicles called exosomes were surface-functionalized and stabilized with polymers to circumvent two major shortfalls associated with biological vesicles, namely, (1) naturally occurring biodegradation, which causes stability problems and (2) high rates of metabolic processing by the body, which results in short blood circulation life-time that is insufficient for adequate targeting of therapeutic sites. Overall, the two highlighted shortfalls have hindered the successful application exosomes as drug delivery nanocarriers, even though exosomes have shown better specificity in functionality relative to polymersomes. WP4. Advanced Characterization: Subject to analysis were the size, aggregation number, encapsulation quantification, degree of hybridization, morphology, shape, scaling properties, compactness, and stability of the novel synthetic and biosynthetic vesicles. These synthetic and biosynthetic vesicles have complex heterogeneity in their molecular properties, and therefore, separation techniques are prerequisite for accurate and reliable characterizations of their molecular properties and the distributions thereof. Moreso, these vesicles are fragile and delicate, and any one of the stipulated molecular parameter is only measurable with channel-based separation techniques such as multidetector FFF, as employed in the USOME action. For visual analysis of microstructure cryo-TEM was utilized. The functionalization and modification to the nanocarriers induced subtle changes in microstructure, which are not detectible with FFF. As such, SANS was employed as a robust and complementary analytical strategy, owing to its better sensitivity to size dynamics.
