Thesis (PH. D)--University of Pennsylvania, 1907. / Bibliography: p. 81-83.

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PEGylation can improve the pharmacokinetic properties of protein therapeutics via decreasing renal clearance and shielding the protein surface from proteases, antibody neutrailization, and aggregation. Conformational stability enhancement can provide criteria for the identification of optimal sites for PEGylation, but how PEG influence the noncovalent interactions from the surface of proteins has not been well illustrated. Macrocyclization can effectively enhance the conformational stability of small peptides and large proteins. Combination of PEG-based conformational stability enhancement and macrocyclization-based conformational constraint has not been explored. Macrocycliziation has been employed to stabilize protein tertiary structures, but there are no general guidelines for interhelical staple to stabilize coiled-coil motifs of proteins. Chapter 1 is an introduction to peptide stapling and macrocyclization of proteins. Chapter 2 describes our test of the hypothesis that PEG increases the conformational stability of proteins by desolvating nearby salt bridges. In chapter 3, we explore the combination of PEG-based conformational stability enhancement with macrocyclization on WW domain, and find that the most important criteria for PEG stapling is ensuring the side chains cross-linked by PEG are distant in primary sequence but close in tertiary structure. In chapter 4, we further apply this macrocyclization criteria to another -sheet-based protein, SH3 domain of the chicken Src protein, and to a disulfide-bonded parallel coiled-coil heterodimer derived from the yeast transcription factor GCN4. In chapter 5, we explore the determinants of PEG-staple-based stabilization by changing the distance of the staple to the terminal interhelical disulfide bond, varying the length of staple, exploring different solvent exposed positions for stapling and employing heterochiral residues for stapling. We further apply the interhelical PEG staple to a HER-2 affibody, and find that PEG-stapling increases the conformational stability and proteolytic resistance of the stapled affibody relative to its non-stapled counterpart and to the native unmodified affibody.

A microencapsulation-based technology platform for effective delivery of multiple micronutrients for food fortification has been developed. The technology, consisting of extrusion agglomeration followed by encapsulation through surface coating, has been successfully tested on three size scales in typical staple foods: as a surface treatment on salt and sugar, on 20-100µm scale; in salt on a 300-1200 μm scale; and on reconstituted rice on the 5-10 mm scale. The process results in effective delivery systems for one or more active ingredients with organoleptic properties that are unnoticeable to the average consumer. Particularly, salt double fortified with iodine and iron using the microencapsulated ferrous fumarate premix made by the extrusion-based agglomeration process had acceptable sensory properties and stability when stored at 40oC and 60% relative humidity (RH) for up to a year. In these tests >85% of iodine and >90% of ferrous iron were retained. Reconstituted Ultra Rice® grains made by extrusion stabilized by internal gelation has resulted in improved grain integrity and a much simplified process, compared to the original, patented surface crosslinking technique. The most effective internal gelation system is composed of alginate, calcium sulphate (CaSO4), and sodium tripolyphosphate (STPP) at a best ratio of 3%:3%:0.6% (w/w). It is feasible to incorporate folic acid into the existing fortification programs using the technology platform developed in this study. The results indicate that the potential interactions of folic acid with other added micronutrients or with the food vehicles could be prevented by incorporating folic acid as a premix made by the extrusion-based technology. Virtually no folic acid was lost after 9 months storage at 40oC and 60% RH when the folic acid premix was added into salt or sugar samples. The technical feasibility of the microencapsulation-based technology platform has been successfully demonstrated for micronutrient delivery in food vehicles of different size ranges, resulting in fortified staple foods with desired physical, chemical, nutritional, and organoleptic properties. The technology should be adaptable to formulating customized delivery systems of active ingredients for broader applications, and promises to bring immediate benefits in combatting micronutrient deficiencies, that will have far reaching effects in health and social development.

A microencapsulation-based technology platform for effective delivery of multiple micronutrients for food fortification has been developed. The technology, consisting of extrusion agglomeration followed by encapsulation through surface coating, has been successfully tested on three size scales in typical staple foods: as a surface treatment on salt and sugar, on 20-100µm scale; in salt on a 300-1200 μm scale; and on reconstituted rice on the 5-10 mm scale. The process results in effective delivery systems for one or more active ingredients with organoleptic properties that are unnoticeable to the average consumer. Particularly, salt double fortified with iodine and iron using the microencapsulated ferrous fumarate premix made by the extrusion-based agglomeration process had acceptable sensory properties and stability when stored at 40oC and 60% relative humidity (RH) for up to a year. In these tests >85% of iodine and >90% of ferrous iron were retained. Reconstituted Ultra Rice® grains made by extrusion stabilized by internal gelation has resulted in improved grain integrity and a much simplified process, compared to the original, patented surface crosslinking technique. The most effective internal gelation system is composed of alginate, calcium sulphate (CaSO4), and sodium tripolyphosphate (STPP) at a best ratio of 3%:3%:0.6% (w/w). It is feasible to incorporate folic acid into the existing fortification programs using the technology platform developed in this study. The results indicate that the potential interactions of folic acid with other added micronutrients or with the food vehicles could be prevented by incorporating folic acid as a premix made by the extrusion-based technology. Virtually no folic acid was lost after 9 months storage at 40oC and 60% RH when the folic acid premix was added into salt or sugar samples. The technical feasibility of the microencapsulation-based technology platform has been successfully demonstrated for micronutrient delivery in food vehicles of different size ranges, resulting in fortified staple foods with desired physical, chemical, nutritional, and organoleptic properties. The technology should be adaptable to formulating customized delivery systems of active ingredients for broader applications, and promises to bring immediate benefits in combatting micronutrient deficiencies, that will have far reaching effects in health and social development.

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