Protein Corona Inhibits Endosomal Escape of Functionalized DNA Nanostructures in Living Cells

Abstract

DNA nanostructures (DNs) can be designed in a controlled and programmable manner, and these structures are increasingly used in a variety of biomedical applications, such as the delivery of therapeutic agents. When exposed to biological liquids, most nanomaterials become covered by a protein corona, which in turn modulates their cellular uptake and the biological response they elicit. However, the interplay between living cells and designed DNs are still not well established. Namely, there are very limited studies that assess protein corona impact on DN biological activity. Here, we analyzed the uptake of functionalized DNs in three distinct hepatic cell lines. Our analysis indicates that cellular uptake is linearly dependent on the cell size. Further, we show that the protein corona determines the endolysosomal vesicle escape efficiency of DNs coated with an endosome escape peptide. Our study offers an important basis for future optimization of DNs as delivery systems for various biomedical applications.

Geometry of a DNA Nanostructure Influences Its Endocytosis: Cellular Study on 2D, 3D, and in Vivo Systems

“…The type of cell line also plays a vital role in the internalization of DNA nanostructures. Smolková et al have demonstrated the effect of cell size in the internalization of DNA nanostructures where they found linear correlation between cell area and uptake . We studied the uptake of DNs in a panel of cell lines originating from different sources and body tissues including SH-SY5Y, HeLa, SUM159, KB3, and RPe1.…”

“…Different fluorophores were excited using different lasers, i.e., for Hoechst 405 nm, Tf-A488:488 nm, TD-Cy3:561 nm, and Gal3-Cy5 633 nm. The image analysis was done using Fiji ImageJ software. ,, The background from each image was subtracted using Fiji ImageJ software (NIH), and whole cell intensity was quantified using maximum intensity projection. The autofluorescence of the cells was eliminated by quantifying the signal from the unlabeled cells.…”

“…Smolkováet al have demonstrated the effect of cell size in the internalization of DNA nanostructures where they found linear correlation between cell area and uptake. 33 We studied the uptake of DNs in a panel of cell lines originating from different sources and body tissues including SH-SY5Y, HeLa, SUM159, KB3, and RPe1. We found 150 nM is the optimum concentration to study the cellular uptake from the previous experiment and was used for further experiments, as at lower concentration some DNs were not showing significant fluorescence signal.…”

Mechanical Regulation of Mitochondrial Dynamics and Function in a 3D-Engineered Liver Tumor Microenvironment

“…For high-quality confocal images and 3D and 4D analysis, we used the brand-new high-resolution spinning disk confocal system IXplore SpinSR (Olympus, Tokyo, Japan). , Cells (HepG2 and Alexander cell lines) were grown in either the monolayer culture (MC) or collagen scaffolds (CSs) under regular culturing conditions, i.e., humidified atmosphere with 5% CO 2 at 37 °C. Afterward, cells were labeled with specific fluorescent probes; see details in Table S2.…”

Uptake and stability of DNA nanostructures in cells: a cross-sectional overview of the current state of the art

“…Predominantly, the DNA tetrahedron (∼10 nm) is used in these studies but over the last several years larger DNA origami structures (∼100 nm) have become more common, likely due to the availability of more sites to couple biocompatible signaling molecules. We observed that the display of targeting antigens or ligands on the surface of the DNA NS is not a requirement for uptake and that DNA NS with 21,31,46,47 and without 5,34,48,49 targeting moieties have been reported to be internalized by cells. The spatial arrangement of antigens on the DNA NS does, however, perform significantly better in immunostimulatory applications since it leverages B/T-cell receptor antigen pattern recognition and macrophage functionality.…”