DNA nanotechnology produces precision nanostructures of defined chemistry. Expanding their use in biomedicine requires designed biomolecular interaction and function. Of topical interest are DNA nanostructures that function as vaccines with potential advantages over non-structured nucleic acids in terms of serum stability and selective interaction with primary human immune cells. Here we describe how compact DNA nanobarrels bind with a 400-fold selectivity via membrane anchors to white blood immune cells over erythrocytes, without affecting cell viability.The selectivity is based on the preference of the cholesterol lipid anchor for the more fluid immune cell membranes compared to the lower membrane fluidity of erythrocytes. Compacting DNA into the nanostructures also gives rise to increased serum stability. The DNA barrels furthermore functionally modulate white blood cells by suppressing the immune response to pro-inflammatory endotoxin lipopolysaccharide. This is likely due to electrostatic or steric blocking of toll-like receptors on white blood cells. Our findings on immune-cell specific DNA nanostructures may be applied for vaccine development, immunomodulatory therapy to suppress septic shock, or the targeting of bioactive substances to immune cells. KEYWORDS DNA; DNA nanotechnology, bilayer membrane, white blood cells, lipids, immunomodulation, nanostructures ToC GRAPHIC 3 DNA nanostructures advance nanotechnology and the life sciences. Compared to other materials, DNA nanostructures have an unsurpassed highly controllable architecture which is based on predictable folding using base-pairing rules. [1][2][3][4][5] By exploiting these properties, the functional structures are increasingly designed to benefit areas outside DNA nanotechnology. Examples include DNA scaffolds which precisely position proteins and other biomolecular components for biophysical and molecular biological research, [1][2][6][7][8] or as scaffold to organize enzymes into efficient multistep biocatysts. 9 Furthermore, predictable changes in DNA nanostructures have been exploited for smart biosensing 10 to measure pH inside cells [10][11][12] or to delivery bioactive cargo into cells. 13 The greatest reward is expected in biomedicine [14][15][16] as illustrated by a DNA nanorobot to deliver anti-cancer drugs 17 or larger nanostructures that mitigate acute kidney injury within animal models. [18][19] Immunology and vaccine development are of topical interests for DNA nanotechnology. Nonstructured DNA and RNA have previously been developed into vaccines against cancer. [20][21][22][23] The relevance of nucleic acids-based therapy platforms has been further increased with the SARS-CoV-2 pandemic. 24-29 A main advantage of mRNA type vaccines is the speed at which they can be developed and manufactured compared to traditional protein-based vaccines. Nevertheless, DNA and RNA vaccines have disadvantages including their low potency, poor uptake into immune cells, lack of stability against nucleases, and fast clearance rates. 20,30-31 These la...

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Mitochondrial dysfunction and immune cell dysfunction are commonplace in sepsis and are associated with increased mortality risk. The short chain fatty acid, butyrate, is known to have anti-inflammatory effects and promote mitochondrial biogenesis. We therefore explored the immunometabolic effects of butyrate in an animal model of sepsis. Isolated healthy human volunteer peripheral mononuclear cells were stimulated with LPS in the presence of absence of butyrate, and released cytokines measured. Male Wistar rats housed in metabolic cages received either intravenous butyrate infusion or placebo commencing 6 h following faecal peritonitis induction. At 24 h, splenocytes were isolated for high-resolution respirometry, and measurement of mitochondrial membrane potential (MMP), reactive oxygen species (mtROS), and intracellular cytokines (TNF alpha, IL-10) using flow cytometry. Isolated splenocytes from septic and septic butyrate treated rats were stimulated with LPS for 18 h and the effects of butyrate on cytokine release assessed. Ex vivo, butyrate (1.8 mM) reduced LPS-induced TNF alpha (p = 0.019) and IL-10 (p = 0.001) release by human PBMCs. In septic animals butyrate infusion reduced the respiratory exchange ratio (p < 0.001), consistent with increased fat metabolism. This was associated with a reduction in cardiac output (p = 0.001), and increased lactate (p = 0.031) compared to placebo-treated septic animals (p < 0.05). Butyrate treatment was associated with a reduction in splenocyte basal respiration (p = 0.077), proton leak (p = 0.022), and non-mitochondrial respiration (p = 0.055), and an increase in MMP (p = 0.007) and mtROS (p = 0.027) compared to untreated septic animals. Splenocyte intracellular cytokines were unaffected by butyrate, although LPS-induced IL-10 release was impaired (p = 0.039). In summary, butyrate supplementation exacerbates myocardial and immune cell mitochondrial dysfunction in a rat model of faecal peritonitis.