Mechanisms facilitating the uptake of carboxyl-polythene glycol-functionalized gold nanoparticles into multicellular spheroids

Abstract

OBJECTIVES : Nanomedicines represent theragnostic alternatives to traditional candidate drugs, with increased targeting and delivery potential due to their size and functional tailorability. Biological activity typically relies on nanomaterials permeating into the intracellular environment, necessitating characterization of uptake and intracellular trafficking pathways. Spheroids' three-dimensional architecture and heterogenous cellular distribution offer an in-vivo-representative platform to assess the biological activity of nanoparticles (NPs). This study aimed to develop an A549 alveolar carcinoma spheroid model as a NP uptake assessment platform for carboxyl-polythene glycol-functionalized gold NPs affording further biological characterization opportunities in nanomedicine.

METHODS : A549 spheroids were generated via the liquid overlay method, and their morphology and viability were assessed for 21 days. Cytotoxicity was assessed via lactate dehydrogenase release. NP uptake was elucidated using uptake pathway inhibition, combined with CytoViva hyperspectral imaging of sectioned spheroids to count internalized NPs.

KEY FINDINGS : Cytotoxicity was absent for all exposure groups. Clathrin-mediated endocytosis was the primary endocytic mechanism (33.5-54.8% of uptake), which may precede lysosomal degradation. Lysosomal membrane permeabilization appears to be a potential downstream application. Low penetration into spheroids (4.5 μm) suggests the failure of NPs to traverse cellular layers in the spheroid.

CONCLUSIONS : Although poor uptake was observed, a multicellular spheroid model of A549 alveolar carcinoma cells was established, allowing for similar future uptake assessment of various NPs.