Fabrication of Protein Scaffold-Nanoparticle Conjugates for Targeted Cancer Therapeutics

Abstract

Targeted nanotherapeutics for cancer aim to preferentially deliver medicine to the tumor, increasing drug localization and reducing negative side effects. In this study, PSb- PEG nanoparticles were specifically targeted to epidermal growth factor receptors (EGFR), often overexpressed in cancer, using protein scaffold ligands known as Centyrins. Both maleimide-thiol and azide-alkyne conjugation chemistries were evaluated. Gel image analysis indicated similar conjugation efficiencies, but azide-alkyne chemistry is preferable for scale-up because it involves one less synthesis step, resists degradation over time, and requires less excess Centyrin to achieve high conjugation efficiency. Based on surface plasmon resonance, Centyrin-conjugated nanoparticles had 17-fold greater avidity for EGFR than free Centyrin at the highest tested ligand density. Fluorescence-activated cell sorting indicated that intracellular uptake plateaus at 1.9 mol% ligand density, reinforcing the literature observation that excessive binding can inhibit endocytosis. In an effort to transition to a biodegradable construct, PS-b-PEG was replaced with PLA-b-PEG. Experiments were conducted to determine the Flash NanoPrecipitation formulation that produces nanoparticles with properties desirable in a cancer nanotherapeutic, namely a diameter between 70 and 90 nm with a size distribution less than 0.2. Three parameters were varied: block copolymer-to-core ratio, solute concentration, and PLA block length. Based on experimental results, MODDE software generated a model to predict hydrodynamic diameter (R\(^{2}\) = 0.58) and size distribution (R\(^{2}\) = 0.81). The predicted optimal formulation was tested and yielded a nanoparticle with a diameter of 97 ± 16 nm and size distribution of 0.2 ± 0.03.