Functionalization of lithium niobate harmonic nanoparticles with anti-EGFR aptamers for active cancer targeting

Nanoparticle (NP)-based systems have emerged as promising candidates for precise and early diagnosis of malignant tumors and enhanced drug delivery to cancer cells and tissues. However, to achieve their full potential, NPs require surface functionalization with targeting agents, allowing for their directed distribution at specific tumor sites. Efficient active targeting requires not only high-affinity ligands, but also robust surface chemistries that preserve ligand accessibility and minimize non-specific interactions in biological media. Aptamers offer attractive properties for NP targeting, including small size, chemical stability and ease of functionalization, yet their performance can be strongly affected by the selected immobilization strategy. Here we report the preparation of anti-epidermal growth factor receptor (EGFR) aptamer-functionalized harmonic NPs (HNPs) and a comparative assessment of four bioconjugation strategies implemented on lithium niobate (LiNbO3, LNO) NP batches: i) amide bond coupling, ii) freeze-assisted strainpromoted azide-alkyne cycloaddition (SPAAC), iii) thiol-mediated substitution and iv) neutravidin-biotin interaction. Starting from a stable silica/peptide coating engineered to introduce surface reactive handles, each route was evaluated using a thorough physicochemical characterization workflow to quantify its impact on colloidal properties and aptamer immobilization, including hydrodynamic size, ζ-potential and stability in relevant buffers/media, as well as quantification of aptamer loading. Aptamer-conjugated LNO NPs resulting from the amide bond coupling strategy were selected for preliminary biological evaluation. In vitro studies in EGFR-overexpressing cancer cells suggested internalization processes based on receptormediated endocytosis, supporting the feasibility of aptamer-guided targeting for enhanced internalization in EGFR positive cells. Overall, this work provides an experimentally grounded comparison of bioconjugation routes for displaying anti-EGFR aptamers on NP surfaces and