Abstract
DNA nanotechnology has yielded remarkable advances in novel composite materials with diverse applications in biomedicine, making DNA nanostructures (DNs) a promising tool for biosensing, drug delivery, cell modulation, and bioimaging. However, for successful translation of DNA nanostructures to real-world applications, it is crucial to understand how they interact with living cells, and the consequences of such interactions. Foreseeing potential clinical translation of DN-based applications, it is imperative to mechanistically understand interactions between DNs and living cells in well-defined and controlled manner.
Studies on the cell uptake and processing of DNs are predominantly done on artificial tissue culture plastics, which do not represent the mechanic cues that cells experience in vivo. Growing number of research indicate that the cellular physiological microenvironment generates mechanical cues which dramatically affect and regulate key cell functions, e.g. growth, survival, apoptosis, differentiation, and morphogenesis. It becomes evident that cellular function and behavior are regulated by the stiffness of the material surrounding the cell. Integrin- and cadherin-mediated interactions pair the physiological microenvironment of adhesive cells with cytoskeleton. Subsequently, mechanical feedbacks adopt the size, composition, structure of the adhesions and the cytoskeleton of cells. In fact, liver stiffness is linked with the progression of fibrosis and liver cancer. Recent literature summarizes that liver stiffness values between 1-5.5 kPa are denoted as normal, whereas liver stiffness in the range of 10-75 kPa is linked to liver cancer. Overall, stiffer extracellular matrix has been associated with accelerated migration, and promotion of proliferation and chemotherapeutic resistance in hepatocellular carcinoma cells. Our group, as well, showed that the individual cell remodeling of liver cancer cells results in a large-scale colony reorganization to fit the lateral forces caused by external constraints [Pharmaceuticals, 2020, 13, 430]. Further, we elaborated that very soft tumor microenvironment (storage modulus with average value G’ ~ 94 Pa) changes cellular size and profoundly slows cellular proliferation in a YAP-mTOR-mediated manner in hepatic cancer cell lines. Recently, we reported that the soft microenvironment upregulates the glycolysis in liver cancer cells. Cells grown in the soft microenvironment exhibit marked mitochondrial depolarization, downregulation of cytochrome c oxidase I in comparison with stiff monolayer cultures [ACS Biomater. Sci. Eng. 2023, 9, 2408]. Our preliminary study on how mechanical signals emanating from the cell’s microenvironment regulate cell behavior proves that it is a step in the right direction and justifies further research in the field of mechanotransduction.
Taken together, those data imply that mechanics of cellular environment affects cell metabolic activity. Therefore, we hypothesized that such metabolic rewiring of cellular activity might be possibly translated towards DNs processing by cells. Although much is known separately about cell responses to their local physical environments and the size/shape dependent cellular uptake of different nanomaterials, the effect of extracellular matrix stiffness on cellular uptake and processing of nanoparticles generally remains rather fragmented. Knowledge in current literature, on how the stiffness of microenvironment modulates uptake and processing of DNs is basically lacking. Thus, we aim to gain deep knowledge about molecular biophysical mechanisms of modulation of DNs uptake and processing by cells constrained by external mechanical forces. We will analyze in detail how DNs are processed within the cell (transport mechanisms, association with organelles, degradation), and responses of the cell to DNs incorporation.
In order to answer such demanding question, we need a synergetic collaborative framework with leading experts in the field of DNA nanotechnology. We have already established fruitful cooperation with the group of Prof. Stephanopoulos, Arizona State University, USA (https://www.stephanopouloslab.com/). We cooperate very productively, that resulted in high-quality common publications. Our intention is to continue this fruitful cooperation and bring it to a higher level. Cooperation with leading research group (the group of Prof. Stephanopoulos) will ultimately increase quality and enhance excellence of Czech science in the field of DNA nanotechnology. We are confident that such cooperation will propel visibility of Czech basic research at international level. Importantly, our research groups complement each other expertise, that makes us confident in successful implementation of the project. This project will provide highly innovative knowledge on quantitative relationships between transmission of mechanical forces to the cellular lumen and subsequent orchestration of DNs uptake and processing.