Carbohydrate Functionalized Gold Nanoparticles Encode Bacterial Targeting and Immunomodulation

Abstract

Carbohydrates play a pivotal role in the maintenance of the structure and function of cells, and their importance is particularly evident during the course of cell development and differentiation. Nevertheless, carbohydrate-protein interactions (CPIs) are feeble; hence, nature uses the multivalent display of carbohydrates (so-called ‘glycocalyx’) to increase the avidity of the carbohydrate interactions. Following the glycocalyx structure, researchers decorated monovalent carbohydrate ligands on multivalent probes such as nanoparticles, polymers, dendrimers, and liposomes to study carbohydrate-carbohydrate and carbohydrate-protein interactions. In my thesis, I have investigated the role of different sizes and shapes of nanoparticles constructed from gold materials and carbohydrate ligands and their applications in bacterial biosensors, immune modulation, and vaccine development. Chapter 1 summarizes different multivalent carbohydrate probes and their major biological and medical applications. More specifically, we address synthetic strategies used for decorating the different types of multivalent systems, ranging from nanoparticles to supramolecular structures of different shapes, to fine tune the spatial arrangement of carbohydrate ligands to improve the avidity of CPIs. Finally, we discuss Kikkeri lab findings on the shape of the nanoparticles’ mediated modulation of carbohydrate-protein interactions. Chapter 2 describes the synthesis of multivalent probes composed of heterogeneous carbohydrate ligands and different topologies of gold nanoprobes for selective targeting and inhibiting bacterial infection. The α-D-mannose and β-D-galactose were successfully conjugated to the homo and heterogenous glycodendrons, followed by functionalizing these ligands on sphere- and rod-shaped gold nanoparticles to generate dual-functional probes. Bacterial binding and inhibition assays were examined in FimH-expressed and knockout E. coli bacteria. Noticeably, the multivalent display and shape of the nanoprobes showed remarkable sensitivity and selectivity for FimH-mannose-mediated E. coli binding. At the same time, the heterogeneity of the dendrons showed the least impact on bacterial adhesion. This trend continued even with the inhibition of bacterial infection of HeLa cells, revealing new insight into the role of gold nanoparticles (AuNPs) shapes and heterogeneity in carbohydrate-protein interactions. Chapter 3 deals with deciphering the size of gold nanoparticles that mediate immune modulation of sialic acid antigens. As a prototype, we synthesized Neu5Gc antigen ubiquitously expressed on human tissues, and interaction with anti-Neu5Gc antibodies enhanced inflammation, which could facilitate tumor progression. In this project, we functionalized Neu5Gc glycan on gold nanoparticles of different sizes and immunized the Cmah-knockout mice. As a control, Neu5Gc glycan was also functionalized on CRM197 protein and immunized with an alum adjuvant. We found that carbohydrates were not able to induce effective immune responses compared to the CRM197 conjugated system. To facilitate better immune responses of Neu5Gc glycans. We have synthesized a tripartite system composed of synthetic TLR7/8 adjuvant, Neu5Gc glycopeptides, and cholesterol moiety. Covalent linkage of these three components is expected to drive the Neu5Gc antigen to the lymph node and modulate T-cell-dependent immune responses. Chapter 4 demonstrate the design and synthesis of CpG-adjuvant coated sphere-, rod-, and star-shaped AuNPs were conjugated to the tripodal Tn-glycopeptide antigen to study their DC uptake and the activation of T-cells in the DCs/T-cell co-culture assay. Our results showed that sphere- and star-shaped AuNPs displayed relatively weak receptor-mediated uptake, endosomal sequestration induced a high level of T helper-1 (Th1) biasing immune responses compared with rod-shaped AuNPs, showing that receptor-mediated uptake and cytokine secretion of nanostructures are two independent mechanisms. Significantly, the shapes of AuNPs and antigen/adjuvant conjugation synergistically work together to modulate the effective anti-Tn-glycopeptide immunoglobulin (IgG) antibody response after in vivo administration of the AuNPs. These results show that by varying the shape parameter, one can alter the immunomodulation, leading to the development of carbohydrate vaccines.