Degree

Bachelor of Science (B.S.) in Biochemistry

Major(s)

Biochemistry

Document Type

Immediate Campus-Only Restricted Access

Abstract

The complement protein C3d plays a crucial role in the innate immune system by opsonizing foreign surfaces, preparing them for phagocytic clearance via macrophage complement receptors. The structure of C3d is characterized, but the influence of its surface orientation on nanoparticle-immune system interactions has not yet been systematically explored. We engineered three cysteine variants of C3d: E167C, Y273C, and P294C. These variants were created to control the protein orientation when conjugated to gold nanoparticle (AuNP) surfaces through specific gold-thiol chemistry. E167C is a disruption right through helix α8 of the CR2- binding patch, Y273C is at the structured C-terminal helical region, and P294C is at a disordered C-terminal tail. Dynamic light scattering and zeta potential measurements confirmed stable and orientation-dependent protein corona formation for all the variants. Circular dichroism spectroscopy verified the native structure was preserved after AuNP conjugation. Thermal denaturation analysis revealed that E167C has been destabilized significantly more than the other variants, consistent with the disruption of the CR2-binding patch. Macrophage uptake experiments in RAW 264.7 cells were quantified by ICP-MS and demonstrated more than a 10- fold orientation-dependent range in intracellular gold accumulation. Y273C-AuNPs had the highest at ~150 ppm, followed by P294C-AuNPs at ~33 ppm, then E167C-AuNPs at ~10 ppm, and lastly bare AuNPs at ~1.5 ppm. These results confirm that C3d surface presentation is a primary determinant of the complement-mediated phagocytosis by macrophages, with direct implication for the design of nanoparticle drug delivery systems.

Date Defended

4-22-2026

Funding Source

This work was supported by the National Science Foundation under awards OIA 2414443, and CBET 2405018.

Thesis Director

Dr. Nicholas Fitzkee

Second Committee Member

Dr. Galen Collins

Third Committee Member

Dr. Christian Flow

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Digital Object Identifier (DOI)

https://doi.org/10.54718/UTIV8423