Acs Nano. unfunctionalized or various functionalized DNA structures to cell lines or even in whole organisms have been demonstrated in a large number of studies (2C13). Interestingly, in the context of drug delivery purposes, the rigid or compact nature of DNA structures appears to make them more prone for entering living cells than linear single- or double-stranded DNA (14). For example, efficient cellular uptake of Spherical Nucleic Acids (SNAs) was demonstrated to be facilitated by scavenger receptors (15,16). Consistently, we observed uptake of a pristine octahedral DNA nanocage in COS-1 cells transfected with a scavenger receptor (Lox1) expressing plasmid but not in un-transfected COS-1 (17) or HEK293T cells (18). The Fan and Tuberfield groups, on the other hand, demonstrated uptake of a pristine DNA tetrahedron in un-transfected HeLa and HEK293T cells (19,20) and studies suggest that the INCB28060 pointy-ends in tetrahedral structures may facilitate internalization (21,22). Once internalized, the cellular stability of DNA structures is rather high varying between 24 and 48 h?depending on the identity of the structure (20,23,24). Unless targeted to other organelles, most DNA INCB28060 structures are directed to the phagolysosomes of treated cells (17,19,23,25,26). This localization presents an obvious advantage, when it comes to treatment of infectious diseases caused by intracellular pathogens that use phagolysosomes or immature phagosomes in macrophages as a safe haven to escape the host immune system and reproduce. Examples of such pathogens are the bacteria and sp. or eukaryotic parasites such as and sp. that all cause severe, in some cases TNFRSF9 deathly, human diseases (27C30). Common for these pathogens is, that they are engulfed by macrophage phagocytosis. Normally, phagosomes fuse with lysosomes and mature into phagolysosomes with an extreme acidic environment that degrade the invading pathogen (31,32). However, pathogens like the above-mentioned species survive by delaying or circumventing the maturation of phagosomes (32). Roughly speaking, pathogens that can survive phagocytosis can be subdivided into three main groups. Members of the first group (group 1) prevent phagosome maturation and includes and sp. (31,32). Members of the second group (group 2), including and were selected as model pathogens from the groups 1 and 2, respectively. A DNA nanoflower (NF) structure originally described by the Tan group was chosen as the DNA structure for our studies (10). NF structures were previously characterized as spherical entities with diameters from 200 nm to 4 m when analyzed by scanning electron microscopy (SEM) (10). NF self assembles from long tandem repeats of DNA generated by Rolling Circle Amplification (RCA) of circular 63- to 123-mer oligonucleotide templates (37). The particular NF used in the current study was generated from a 97-mer template. Although NFs are characterized by a higher degree of heterogeneity than tile- or origami-based DNA structures, NFs can be produced with relatively well-defined sizes and structures (11,38,39). Of particular interest for real-life applications, INCB28060 NF present considerable advantages associated to the ease by which they can be functionalized, labeled and produced in high quantities from a single template and primer oligonucleotide in a simple one-step procedure at reasonably low costs. In the current study we successfully demonstrate specific uptake of a fluorescently labeled NF in macrophages, accumulation of the NF in phagolysosomes and co-localization with both or in infected cells. This illustrates the potential of employing such DNA structure as a carrier for specific targeted treatment of diseases such as.