CRISPR Cas Proteins Coronated AuNP Nanostructure for Enhanced Uptake Efficiency into Cells

Abstract

Nanotechnologies hold significant promise for biosensing and biomedical applications; however, their effectiveness is often limited by the unstable accumulation of nanoparticles in biological environments. In this study, we designed and characterized an artificial protein coronated nanostructure (PCN) to improve intracellular delivery. Gold nanoparticles (AuNPs) were functionalized with thiol-modified DNA strands complementary to a DNA linker capable of hybridizing with single-guide RNA (sgRNA). The Cas9 protein binds to sgRNA, forming a uniform and controllable protein corona nanostructure on the nanoparticle surface. Stability tests of the PCN under serum and varying concentrations of glutathione demonstrated its robustness and adaptability in complex biological matrix. Using confocal fluorescence microscopy with fluorophore-labeled PCNs and inductively coupled plasma mass spectrometry (ICP-MS) for gold quantification, we confirmed that PCNs exhibit significantly enhanced intracellular delivery efficiency and accumulation compared to conventional spherical nucleic acids (SNA). Mechanistic studies revealed that this improvement is primarily mediated by clathrin-dependent endocytosis, with additional involvement of caveolae-mediated pathways. This biocompatible PCN-based delivery platform not only mitigates non-specific nanoparticle aggregation but also significantly improves cellular uptake efficiency, making it a promising strategy for imaging and biomedical applications.