EGFR Ligand Clustering on E2 Bionanoparticles for Targeted Delivery of Chemotherapeutics to Breast Cancer Cells

Abstract

Naturally occurring protein nanocages are promising drug carriers because of their uniform size and biocompatibility. Engineering efforts have enhanced the delivery properties of nanocages, but cell specificity and high drug loading remain major challenges. Herein, we fused the SpyTag peptide to the surface of engineered E2 nanocages to enable tunable nanocage decoration and effective E2 cell targeting using a variety of SpyCatcher (SC) fusion proteins. Additionally, the core of the E2 nanocage incorporated four phenylalanine mutations previously shown to allow hydrophobic loading of doxorubicin and pH-responsive release in acidic environments. We functionalized the surface of the nanocage with a highly cell-specific epidermal growth factor receptor (EGFR)-targeting protein conjugate, 4GE11-mCherry-SC, developed previously in our laboratories by employing unnatural amino acid (UAA) protein engineering chemistries. Herein, we demonstrated the benefits of this engineered protein nanocage construct for efficient drug loading, with a straightforward method for removal of the unloaded drug through elastin-like polypeptide-mediated inverse transition cycling. Additionally, we demonstrated approximately 3-fold higher doxorubicin internalization in inflammatory breast cancer cells compared to healthy breast epithelial cells, leading to targeted cell death at concentrations below the IC50 of free doxorubicin. Collectively, these results demonstrated the versatility of our UAA-based EGFR-targeting protein construct to deliver a variety of cargoes efficiently, including engineered E2 nanocages capable of site-specific functionalization and doxorubicin loading.

Genetically Encoded Self-Assembling Protein Nanoparticles for the Targeted Delivery In Vitro and In Vivo

“…Thus, the cells were pre-targeted with GE11-mCherry-SpyCatcher fusion protein and then labeled with E2 nanoparticles fused with SpyTag with subsequent confocal microscopy study. The particles were loaded with doxorubicin and the cytotoxicity tests confirmed that doxorubicin loading into the nanoparticles is more efficient than the delivery of free doxorubicin [ 152 ].…”

Highly modular hepatitis B virus-like nanocarriers for therapeutic protein encapsulation and targeted delivery to triple negative breast cancer cells

“…Nanoparticle delivery platforms improve drug biodistribution and pharmacokinetics and can be modified with targeting moieties for cell-specific uptake. [12][13][14][15] Nanoparticle encapsulation provides the simultaneous delivery of multiple copies of a target protein cargo, which enhances the effective concentration at the delivery site. However, conventional liposomal and polymeric nanoparticles typically require non-aqueous synthesis conditions that may affect cargo protein stability, and they are often polydisperse in size and potentially toxic, yielding slow and nonuniform drug release and off-target cytotoxicity.…”

Multivalent Ligand-Nanoparticle Conjugates Amplify Reactive Oxygen Species Second Messenger Generation and Enhance Epidermal Growth Factor Receptor Phosphorylation

“…Multivalent ligand presentation is an important design parameter of nanoparticles (NPs) intended for therapeutic − or analytical applications, as it enhances the interactions between NPs and cellular targets. The multivalent display of ligands on NPs leads to increased binding avidity − to the corresponding cell surface receptors with the potential to elicit different cellular responses due to changes in targeting efficiency, − uptake, − and intracellular trafficking. − Importantly, multivalent ligand–receptor interactions on NPs promote receptor clustering, − which can further modulate receptor function and activation. − The mechanisms of this multivalent receptor modulation and how they can be controlled through rationally designed NPs still raises important questions.…”