Nanoparticles and the DNA Corona: Biophysical Characterization and Immunological Implications

Abstract

Autoimmune diseases are highly complex and heterogeneous in their manifestations. DNA has been implicated as an important antigen and biomarker in a range of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease and diseases such as cancer. Cell-free DNA is released when cells undergo apoptosis or necrosis and the size of properties of the released DNA is dependent on how cells die. Studies have shown that a significant portion of cell-free DNA exists inside extracellular vesicles such as exosomes or microparticles. Elevated microparticle numbers have been implicated in the pathogenesis of systemic lupus erythematosus as a source of DNA autoantigen. Furthermore, immune complexes consisting of anti-DNA antibodies bound to the surface or microparticles has been seen in patients with lupus. This finding suggests that DNA is not only present inside particles but is immunologically assessable due to surface adsorption. Isolating microparticles can be difficult due to the complex biological environment in which they are present, Furthermore, due to their heterogeneity of particles, studying specific features of these particles with DNA on the surface presents a major challenge. In this work, we use a model system to study the interaction of DNA and particles with immune cells. We use synthetic polystyrene particles ranging in size from 40 nm – 10 µm, E.coli and calf-thymus DNA of various lengths, and mouse and human macrophages to better understand the biophysical properties of the DNA/particle interaction as well as the innate immune response to these DNA-particle complexes. This dissertation begins by characterizing the adsorption of E.coli DNA onto nanoparticles of size 200 nm and 2 µm. We study how the degradation of DNA by DNase 1 is impacted due to adsorption on particles vs when DNA exists free in solution and show that particles offer some degree of protection. We then use murine macrophages to show that DNA that is particle adsorbed is significantly more stimulatory than free DNA due to an increased production of TNF and IL-6 by the cells which are key inflammatory cytokines associated with lupus. We explore two DNA sensing pathways in the macrophage cells: TLR9 and cGAS-STING and show that the cGAS-STING pathway is activated when cells interact with DNA-particles but that when cells interact with free DNA, TLR9 and cGAS-STING are activated. To further explore the differences in macrophage stimulation between DNA-particles vs DNA or particles alone, we ran an RNA-sequencing analysis to see how the overall transcriptomic profile changes when cells are exposed to these treatments. In this study, to bolster the biological significance, we used human macrophage like cells and calf-thymus DNA. Mammalian DNA is inherently non-stimulatory alone so this would allow us to better highlight specific upregulated genes of interest when DNA is on particles. Our analysis revealed that there is a large change in gene expression for these cells that are treated with DNA-particles vs either alone. We found that the number of differentially expressed genes is much higher (> 300) when cells are treated with DNA-particles vs (< 40) cells treated with bare particles and (0) for cells exposed to free DNA. Furthermore, we observed that among the significant genes, many of them were cytokines and chemokines that upregulated as part of an inflammatory response. When we looked at significantly enriched gene ontologies, we again saw that for cells treated with DNA-particles, the most significant gene ontologies that were enriched were related to cytokine activity and inflammation. Finally, we wanted to further explore the biophysical characteristics of the DNA-particle interaction. We used particles ranging in size from 40 nm – 10 µm as well as planar surfaces. We used 2 lengths of DNA (DNAS ~500 bps and DNAL~10,000), as well as single stranded DNA. We also wanted to see if particles incubated with a stoichiometric vs excess amount of initial DNA changes the interaction. We found that as size of NPs increase, we get increased DNA adsorption due to the larger surface area with more DNA adsorption when particles are incubated with excess DNA. We looked at the differences in DNA degradation by DNase 1 and found that particles at the very small end (40 nm) offered more protection, with a decrease as particle size increased to 200 nm, and then protection increased again as particles got even larger to 1, 2 and 10 µm. When we looked at single stranded DNA, this protective effect was completely revered with 200 nm particles offering the most protection.In summary, this dissertation outlines the biophysical interaction of DNA and particles as well as the DNA-particle stimulatory effect of immune cells. The work here was done to better understand how biological microparticles interact with DNA in circulation as these complexes have deep ties with autoimmune disease. A better understanding of these systems will hopefully allow for the development of better diagnostics and therapies, and we are excited for our work to have an impact across an interdisciplinary range of fields including immunology, biophysics, and materials science.