Development of Gold Nanoparticles for Targeting PD-L1-Presenting Breast Cancers
Date
2023-07-31
Authors
Blevins, Derek James
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Abstract
Our goal was to selectively target the immunosuppressive programmed cell death protein ligand 1 (PD-L1) presenting cancer cells with gold nanoparticles (AuNP). Initially, small molecules, described as antagonists for the PD-1/PD-L1 interaction, were considered as promising targeting agents for the gold platform. The competitive binding profiles of these molecules were evaluated using surface plasmon resonance (SPR). However, we found that none of the small molecule candidates were capable of disrupting the interaction, despite their apparent cell-based efficacy in other literature. Our findings indicate that the molecules were being mischaracterized as immunomodulators directly blockading the PD-1/PD-L1 interaction. In addition, we found no evidence of direct binding with the small molecules to either PD-1 or PD-L1, indicating they would not be suitable candidates as targeting agents. Instead, a commercially available monoclonal antibody (mAb, αPD-L1; from BioXCell) was found as a suitable alternative for targeting and blockading PD-L1 directly from PD-1.
The antibody was conjugated with α-lipoic acid (αLA) through its NHS-ester such that the thiolated mAb may be grafted to functionalize the gold cores. The degree of functionalization on the gold core was quantified in vitro using SPR, where the relationship between valency and molecular weights of the gold core and unbound mAb binding to PD-L1 was studied.
This SPR method was primarily used further to optimize the AuNP formulations until they were deemed suitable for cellular work. Gold cores with a surface area coated in 25% αLA-mAb and 75% PEG2000 had shown a near maximal response of binding relative to the unbound mAb, indicating a high degree of functionalized nanoparticles. This formulation was then moved forward into cellular work with naïve human white blood cells (Jurkat) that were stimulated with PHA (to produce PD-1), and the stimulation suppressed by the presence of PD-L1.
In addition to AuNP formulation, we sought to investigate controlled release mechanisms indicative of the reducing character of the tumour microenvironment. We developed a series of disulfide tethers with a fluorogenic dye that induces turn-on fluorescence upon disulfide exchange, expected to show selectivity for the higher concentration of glutathione. Instead, we found that the common, but necessary, additive, fetal bovine serum (FBS), was triggering the premature release of our tethered fluorophore, disrupting our intended controlled-release studies. Through systematic investigation, we found that unwanted turn-on fluorescence from our dithiodiacid tethers was ultimately occurring due to esterase activity found in the FBS. This activity was shut down when methyl groups were installed at the α-position to the carbonyl carbon. Future work may use these bulkier dithiodiacid tethers for selective release in other AuNP formulations.
Immunotherapeutic efficacy of the functionalized AuNPs was investigated by showing consistent stimulation in the presence of immunosuppressive PD-L1. Naïve Jurkat cells were stimulated with phytohemagglutinin (PHA), where the cell density would significantly increase relative to basal cell growth, and in the presence of solubilized PD-L1, show no change in density attributed to T cell exhaustion. When AuNPs were present, the cell density would reflect that of “uninhibited” stimulation control, regardless of PD-L1 presence, indicating the potential immunotherapeutic benefit of recovery from immune exhaustion.
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Keywords
surface plasmon resonance (SPR), gold nanoparticles, Ligand binding, immunostimulation, immunotherapy, in vitro studies, assay development, SPR, AuNP