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Date of Award

2016

Document Type

Campus Access Dissertation

Degree Name

Doctor of Philosophy in Biochemistry and Molecular Biology (PhD)

Administrative Home Department

Department of Chemical Engineering

Advisor 1

Ching-An Peng

Committee Member 1

Ashutosh Tiwari

Committee Member 2

Rupali Datta

Committee Member 3

Lanrong Bi

Abstract

Localizing the drug at the site of action is one of the most important goals of drug delivery systems. This requires developing a vehicle which is able to protect drug carriers from degradation, while delivering the drug of interest to the specific tissue. In this study, a novel recombinant protein RGD-CD47-Streptavidin was synthesized to modify the surface of the drug carriers, and thereby allow them to surpass the mononuclear phagocyte system and deliver the drug of interest directly to target cancer cells. The recombinant protein was made and purified using a unique solution containing low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer. The purified protein was characterized by the western blot analysis and its tumor tropic and antiphagocytic functionality was confirmed by the cell assay.

Non-viral and viral drug carries were both functionalized with the recombinant multifunctional protein. As a model of non-viral drug carriers, 1.5107 biotin coated polystyrene particles (150, 560 and 840 nm) were coated with various concentrations of the recombinant fusion protein. Flow cytometry and J774A.1 macrophage cell studies revealed that CD47 immobilized on the various size of particles significantly reduced phagocytosis for 8 hours. However, the soluble CD47 decreased the phagocytes’ particle engulfment just for 2 hours. The phagocytosis index was decreased by increasing the CD47 density on the particle surface. The results demonstrated that presence of 116.49, 45.33 and 287.58 µg/µm2 of CD47-SA respectively on the surface of 840, 560 and 150 nm particles, decreased 50% of phagocytosis after 4 hours. To functionalize viral vectors with the multifunctional protein, lentivirus encoding eGFP was obtained from 293T producer cells and biotinylated by host-cell-assisted labeling strategy. The biotinylated lentivirus was coated with recombinant fusion protein RGD-CD47-SA and characterized by immunoblot analysis. The antiphagocytosis cell study and flow cytometry analysis demonstrated that CD47 self-marker protein potentially inhibited lentivirus uptake by macrophages and therefore decreased infectivity of the virus in comparison with non-CD47 coated virus.

To examine the tumor targeting of the protein, the 560 nm polystyrene particles were coated with CD47-SA and RGD-CD47-SA recombinant fusion protein separately. The coated particles were able to escape from macrophage engulfment by 8 hours. The association of coated particles with integrin αvβ3 was demonstrated in the highly expressed integrin colorectal cancer cells (HT29) and adenocarcinomic human alveolar basal epithelial cells (A549). The polystyrene particles coated with CD47-SA were able to bind to the integrin αvβ3 expressed on the surface of the A549 and HT29 cells but not stronger than the particles coated with RGD-CD47-SA. After blocking the integrin αvβ3, the cellular uptake of functionalized nanoparticles with CD47 and RGD-CD47 became equivalent to the bare nanoparticles showed that the cellular uptake of RGD-CD47 coated nanoparticles was mediated by integrin αvβ3 and RGD was able to boost the tumor targeting of the particles. In summary, the viral and non-viral carriers (size range 100-840 nm) tagged with RGD-CD47 through coreSA-biotin affinity were able to evade phagocytosis and target tumor cells in-vitro.

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