Engineered-affibody conjugates contribute to the specific targeting and cellular retention of polyplexes in Erbb3 overexpressed lung cancer cells

Ligand-modified nanoparticles have shown the ability to specifically bind to tumor cells, improving retention in tumors after initial accumulation driven by the enhanced permeability and retention effect. These particles are typically engineered to bind to receptors overexpressed in cancer cells compared to healthy cells, such as the HER3 (Erbb3) receptor in lung cancer. In this study, we confirmed the overexpression of Erbb3 in various KRAS mutant lung cancer cell lines. An engineered affibody, well-established in previous research, was selected to target Erbb3 as a proof of concept. The affibody was integrated into the particle system via two distinct strategies. In the pre-functionalization approach, the affibody was conjugated to PEI or C14-PEI using SPDP as a linker. A spectral shift technique was then used to assess the affinity of the affibody and affibody conjugates toward Erbb3, allowing us to estimate the half-maximal effective concentration (EC₅₀). Following synthesis and characterization, various polyplex formulations were prepared, including mRNA complexes with PEI-affibody, C14-PEI/PEI-affibody, and C14-PEI/C14-PEI-affibody. In the post-functionalization approach, polyplex formulations composed of different blends of C14-PEI and functionalized Azido-PEI were initially prepared and subsequently modified with DBCO-functionalized affibody via click chemistry. These formulations were prepared at various nitrogen to phosphate (N/P) ratios and characterized in terms of particle size, polydispersity index (PDI), and zeta potential. We also evaluated cellular uptake and eGFP mRNA expression to understand how the different formulations and conjugates influenced ligand-modified polyplex properties and delivery behavior. Our results demonstrated that affibody conjugates can specifically target Erbb3 and promote polyplex accumulation in KRAS-mutated lung cancer cells. We further analyzed the impact of conjugation methods and affibody density on polyplex design and performance. In conclusion, this study highlights the advantages of using specific targeting ligands. By optimizing formulation components, conjugation methods, and ligand density, various targeting ligands can be attached to polyplexes, enhancing cell-specific targeting, internalization, and retention. These findings provide valuable insights and a foundation for future targeted therapies and polyplex design.