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Hydrogel microparticles (HMPs) are powerful tools to study and manipulate cellular behavior in 3D cell culture systems and animal models. Here, fully DNA-based HMPs are presented, whose material properties can be precisely tuned by sequence-programmable design of self-assembling DNA nanostructures. These DNA-HMPs offer control over size, stiffness, viscoelasticity and ligand presentation. They are formed by microfluidic encapsulation of two types of orthogonal DNA nanostars and a sequence-complementary DNA linker in water-in-oil droplets. By varying the valency of the DNA nanostar designs, tunable mechanical properties are achieved - spanning three orders of magnitude in Youngs modulus from 30 Pa to 6.5 kPa with distinct viscoelastic behavior. Click-chemistry based functionalization with the small fibronectin-derived peptide cyclic-RGD (c[RGD]) enables integration into fibroblast spheroids. DNA-HMPs are stably retained within the spheroids for several days and undergo design- and stiffness-dependent remodeling, indicating active interactions between the cells and the DNA-HMPs. Combining tunable material properties and inherent biocompatibility of DNA with straightforward functionalization and stimuli-responsiveness, these DNA-HMPs represent a versatile tool to probe and manipulate tissue behaviors in 3D cell cultures and in vivo models. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/665473v2_ufig1.gif" ALT="Figure 1000"> View larger version (51K): org.highwire.dtl.DTLVardef@c226ceorg.highwire.dtl.DTLVardef@81c3b3org.highwire.dtl.DTLVardef@1573ee1org.highwire.dtl.DTLVardef@e1518c_HPS_FORMAT_FIGEXP M_FIG DNA hydrogel microparticles are designed to exhibit controllable viscoelasticity and stiffness across three orders of magnitude from 30 Pa to 6.5 kPa. They are uptaken into fibroblast spheroids where they are actively remodeled by cellular forces depending on their mechanical properties. C_FIG

Scaffolded DNA origami has become a valuable nanoscale tool for applications in biomedical and physical sciences. Critical to leveraging the modular and programmable properties of DNA origami nanodevices is access to the scaffold strand, a long single-stranded DNA (ssDNA) of precise length and sequence, which is folded into a compact shape via piecewise base-pairing with many staple strands, short ssDNA oligonucleotides. Current methods to produce and manipulate long ssDNA scaffolds can be costly, time-consuming, and cumbersome. In contrast, methods to produce and manipulate the sequence of double-stranded DNA (dsDNA) are efficient and scalable. Here, we present a method for the rapid isolation of target ssDNA sequences from a variety of dsDNA sources using oligonucleotides as blocking strands that bind continuously to the undesired strand, thereby releasing the target scaffold strand. We report successful ssDNA isolation from linear and supercoiled dsDNAs of various sequences and lengths, ranging from 769 to 15,101 nucleotides. In addition to isolating ssDNA, we demonstrated this approach enables folding of DNA origami directly from dsDNA templates using both blocking and staple strands in a single-pot thermally controlled reaction. Furthermore, we explore multi-scaffold and gene-encoding DNA origami structures, expanding the framework for application-based designs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/709872v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1cc75dcorg.highwire.dtl.DTLVardef@4df8e2org.highwire.dtl.DTLVardef@10ed113org.highwire.dtl.DTLVardef@1c05bdd_HPS_FORMAT_FIGEXP M_FIG C_FIG

The extrinsic activation of programmed cell death by FasR/CD95 is a promising minimally invasive strategy for cancer treatment. This can be leveraged using high-precision nanoscale therapeutics: Utilizing DNA origami for precise Fas ligand (FasL) presentation resulted in over 100 times more potent apoptosis induction in single cells. However, treating large, solid tumors poses challenges for DNA origami-based therapeutics, including drug distribution and altered cellular behavior. Here, we addressed these challenges using a 3D spheroid model. First, we assessed DNA origamis ability to penetrate tumor tissue, finding that penetration is influenced by the DNA origami size rather its structural flexibility. Second, we evaluated the apoptosis induction efficacy by DNA origami-FasL nanoagents within the spheroid model. The most potent nanoagents were able to completely eradicate all cells in the spheroid. Results indicated that apoptosis induction depended strongly on FasL attachment strategy rather than DNA origami design. Notably, only a rigid neutravidin linker for FasL attachment, rather than a flexible dsDNA linker, halted spheroid growth and fully eradicated all cancer cells. This study offers critical insights into designing DNA-based therapeutics for complex cellular environments and significantly advances DNA origami nanotherapeutic development, highlighting the impact of nanoagent design on cell fate decisions.

DNA base pairs can both encode biological information and be used as a programmable material to build nanostructures with potential application in nanofabrication, data processing and storage, biosensing and drug delivery. Over several decades development of these DNA origami nanostructures has led to increasingly advanced self-assembling nanostructures and molecular machines actuated by various mechanisms such as toehold-mediated strand displacement (TMSD), magnetism and even light. However, scalability remains challenging as using larger scaffold strands can increase the likelihood of kinetic traps and misfolded conformations. Here we describe a repeatable DNA nanohinge system to increase the scalability of existing nanohinge designs for hierarchical assembly of more complex structures with greater degrees of mobility and functionality. The components of this system, comprising two distinct nanohinges, were designed in caDNAno. Structure conformation and stability were simulated using CanDo and MrDNA, and hinge assembly was validated by TEM. Electron micrographs revealed hinge-shaped nanostructures capable of self-assembly into more complex structures, as well as actuation using TMSD through a reversible locking mechanism incorporated into the design. Our work expands the existing utility of DNA nanohinges as building blocks for scalable DNA nanostructures and demonstrates the feasibility of polymerizing hinges in a novel manner for higher order assembly. The enhanced functionality of our dual hinge systems can be employed in future applications requiring greater control and mobility of DNA nanostructures.

A PCR- and sequencing-free mutation detection assay facilitates cancer diagnosis and reduces over-reliance on specialized equipment. This benefit was highlighted during the pandemic when high demand for viral nucleic acid testing often sidelined mutation analysis. This shift led to substantial challenges for patients on targeted therapy in tracking mutations. Here, we report a 30-minute DNA mutation detection technique using Cas12a-loaded liposomes in a microplate reader, a fundamental laboratory tool. CRISPR-Cas12a complex and fluorescence-quenching (FQ) probes are introduced into tumor-derived extracellular vesicles (EV) through membrane fusion. When CRISPR-RNA hybridizes with the DNA target, activated Cas12a can trans-cleave FQ probes, resulting in fluorescence signals for the quantification of DNA mutation. Future advancements in multiplex and high-throughput mutation detection using this assay will streamline self-diagnosis and treatment monitoring at home.

DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.

Lipophilic dyes are widely used to track extracellular vesicles (EVs), yet their labeling efficiency toward bona fide small EVs (sEVs) remains poorly defined. Here, we critically reassess this efficiency using a serum-free HEK293F system that generates endogenously fluorescent protein-tagged sEVs (sEVs-FPT) as an unambiguous positive reference, thereby minimizing interference from co-isolated, dye-labelable non-vesicular extracellular particles (NVEPs). Two orthogonal methods, nanoflow cytometry and fluorescence microscopy, were employed for cross-validation. We found that PKH26, PKH67, and DiD labeled <0.5% of sEVs-FPT, regardless of vesicle heterogeneity. In vivo tracking confirmed that dye-derived signals were far weaker than FPT signals and strikingly failed to colocalize with them. Preliminary mechanistic evidence indicates that this failure is due to an inability of sEVs to actively internalize dye aggregates. Our findings raise serious concerns about the validity of lipophilic dye-based EV tracking and call for a critical reevaluation of the relevant literature.

The mimicry of living tissues from artificial cells is beneficial to understanding the interaction mechanism among cells, as well as holding great potentials in the tissue engineering field. Self-powered artificial cells capable of reversible deformation are developed by encapsulating living mitochondria, actin proteins, and methylcellulose. Upon the addition of pyruvate molecules, the mitochondria produce ATP molecules as energy sources to trigger the polymerization of actin. ATP molecules were produced by mitochondria (2.76x1010/ml) with the concentrations of 35.8{+/-}3.2 {micro}M, 158.2{+/-}19.3 {micro}M and 200.7{+/-}20.1 M by adding pyruvate molecules with the concentration of 3 M, 12 M and 21 M, respectively. The reversible deformation of artificial cells is experienced with spindle shape resulting from the polymerization of actins to form filaments adjacent to the lipid bilayer, subsequently back to spherical shape resulting from the depolymerization of actin filaments upon laser irradiations. The linear colonies composed of these artificial cells exhibit collective contraction and relaxation behavior to mimic muscle tissues. At the stage of maximum contraction, the long axis of each GUV is in parallel to each other. All colonies are synchronized in the contraction phase. The deformation of each GUV in the colonies is influenced by its adjacent GUVs. The muscle-like artificial cell colonies paved the path to develop sustainably self-powered artificial tissues in the field of tissue engineering.

In vitro fertilization (IVF) is essential for many couples facing infertility, e.g. in cases of low sperm count (oligospermia), where natural fertilization is unlikely. Medical microrobotics, making use of microscopic devices designed to perform targeted tasks inside the body under imaging guidance and controlled actuation, represents a promising strategy to guide sperm cells toward the oocyte. This approach may significantly reduce the time, invasiveness, and patient burden of conventional IVF, with long-term potential for in vivo assisted reproduction. Here, we report the first successful in vitro fertilization (IVF) using magnetically functionalized spermatozoa, termed magnetotactic sperm cells (MSCs), as a step toward in vivo microrobotic guidance of sperm cells for targeted artificial insemination. We present a protocol for the preparation of MSCs for their use in IVF, resulting in samples largely free of non-functionalized sperm cells (99.69% purity). We systematically evaluate the effect of particle functionalization on sperm health, including acrosome integrity, DNA fragmentation, mitochondrial membrane potential, oxidative stress, and epithelial interactions, and observe no adverse effects. Notably, MSCs showed improved mitochondrial membrane integrity compared to the control samples after two hours of incubation. Using MSCs, we successfully performed complete IVF cycles that resulted in embryos developing to the blastocyst stage at a comparable rate as non-functionalized sperm cells of the same concentration. Lower concentrations of non-functionalized sperm cells (comparable to those remaining in the MSC sample after purification) did not result in any development of embryos to blastocysts. To facilitate manipulation and translation, we implemented automated image-based recognition, magnetic manipulation, and pre-clustering routines that increased guidance efficiency and are compatible with standard IVF workflows. Together, these results demonstrate that magnetic functionalization can be applied without compromising key sperm quality metrics and can enable directed sperm guidance for assisted oocyte fertilization. This work provides a practical framework for integrating microrobotic sperm manipulation into assisted-reproduction workflows and supports further development toward automated in vitro and eventual in vivo applications.

Extracellular vesicles (EVs) are heterogenous lipid bound membranous structures released by different cells, showing a great potential to be used as biomarkers. They have also been explored for their role in the context of environmental toxicity. When endothelial cells are exposed to pollutants like Polycyclic Aromatic Hydrocarbons (PAH) - the most common being benzo[a]pyrene (B[a]P) - EVs released from those cells undergo surface and cargo modifications. Subpopulations of large EVs (lEVs) have shown to contain either damaged or intact mitochondria which is inexorably linked to oxidative stress conditions. In this paper, we studied B[a]P induced modifications in lEVs derived from endothelial cells, through morpho mechanical characterization with atomic force microscopy (AFM). Colocalizing AFM with fluorescence microscopy allowed us to differentiate between EVs containing mitochondria and those that did not. EVs containing mitochondria had a larger size (maximum diameter) when coming from treated cells (1.8 {+/-} 0.89 {micro}m) as compared to control cells (1.63 {+/-} 0.76 {micro}m). Moreover, their Youngs moduli were higher in the treated condition (3.09 {+/-} 2.54 MPa in average) as compared to the control condition (1.25 {+/-} 0.92 MPa in average). We also observed a heterogeneity within single vesicles, with most Youngs modulus values ranging from 0.1 up to 30 MPa for the treated condition and from 0.1 to 5 MPa for the control condition. Finally, applying linear discriminant analysis (LDA) and Random Forest (RF) algorithms on maximum diameter, height, and distribution of Youngs modulus values, we demonstrated the possibility to discriminate between EV subpopulations. Indeed, we successfully managed to a) distinguish EVs containing mitochondria from the "empty" ones, with an accuracy of 84% and b) discriminate whether these mitochondria-containing EVs originated from control or treated conditions, with an accuracy of 76%. These findings highlight the power of combining morpho-mechanical analysis and machine learning for identifying and discriminating EV subpopulations, no longer requiring any EVs fluorescence labelling.

Negatively charged DNA nanostructures, such as tetrahedral nanocages, are internalized by cells despite the electrostatic repulsion from the anionic cell membrane, and, paradoxically, cancer cells, which carry intrinsically higher negative charge due to overexpression of sialic acids on their cell surface, show markedly higher uptake than normal cells. This contradiction exposes a fundamental gap in our understanding of how these anionic nanostructures overcome this repulsion. Using chemical modulation of cell-surface sialylation in RPE1 cells to create three groups with altered sialylation levels, together with inhibitor-based dissection of endocytic pathways, we demonstrate that an increase in cell surface sialylation governs the uptake of DNA tetrahedra not through electrostatics but by structurally remodeling the cell membrane via rearrangement of the GM1 lipid raft microdomain, recruiting caveolae-mediated endocytosis as an additional pathway alongside clathrin-mediated endocytosis, thereby increasing the intake of the nanostructure. These findings reframe tumor hyper-sialylation as a determinant of the uptake of anionic nanostructures, such as DNA tetrahedra, and as a targetable parameter for rational optimization of DNA-based nanotherapeutics against cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/722926v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@10eede7org.highwire.dtl.DTLVardef@124dd56org.highwire.dtl.DTLVardef@13f5355org.highwire.dtl.DTLVardef@780ecf_HPS_FORMAT_FIGEXP M_FIG C_FIG

DNA-encoded library (DEL) technology enables high-throughput small-molecule discovery but is typically performed using purified proteins under in vitro conditions that do not reflect native intracellular environments. Here, we present a microfluidic agarose -droplet platform for cellular-context DEL screening. The porous hydrogel droplets provide mechanically stable yet permeable microenvironments that protect weak protein-ligand interactions while enabling efficient buffer exchange and ligand diffusion. Importantly, mild cell permeabilization within droplets selectively retained chromatin-associated proteins, allowing screening directly in a cellular context. Using BRD4 as a model target, we validated intracellular ligand engagement by fluorescence imaging and super-resolution microscopy. Small-scale DEL screening selectively enriched JQ1 in both bead-based and cell-based formats, and large-scale DEL screening across millions of encoded compounds successfully identified hit molecules by sequencing. This agarose -droplet-based strategy expands DEL technology toward biologically relevant and chromatin-associated targets under near-native conditions.

Preparation of spermatozoa with optimal developmental competence remains a challenge in assisted reproduction. Conventional techniques based on sperm motility and morphology fail to adequately remove sperm with DNA damage. Here, we report development of a microfluidic device with a functionalized surface, inspired by the physiological processes of immune cell-mediated sperm selection in the female reproductive tract. A plasma-polymerized polyoxazoline (PPOx) film is applied to glass channel slides by deposition of 2-methyl-2-oxazoline, to establish a stable, biocompatible interface confirmed by X-ray Photoelectron Spectroscopy (XPS), ellipsometry, and sperm culture assays. To selectively eliminate pre-apoptotic and apoptotic spermatozoa wherein DNA damage is common, anti-phosphatidylserine (Anti-PS) antibody is immobilized to the PPOx-coated surface proximal to the channel slide inlet, while the sperm chemoattractant progesterone is adsorbed near the outlet. To optimise selective functionality, the surface topography is tailored by covalent immobilization of gold nanoparticles and addition of microchannels. Sperm recovered after processing whole liquified semen then consistently exhibit high motility and morphology, with <1% showing apoptosis-associated membrane damage or DNA fragmentation. Compared with conventional swim-up or other microfluidic approaches, the device yields sperm with improved quality, offering a simple one-step sperm selection strategy with potential for application in human and animal assisted reproduction. Short text and graphic for 45 the Table of Contents (ToC)This study reports a microfluidic device with a functionalized surface utilizing a polyoxazoline coating and covalently immobilized gold nanoparticles and anti-phosphatidylserine antibody. The device selectively eliminates pre-apoptotic and apoptotic spermatozoa and yields sperm with substantially improved quality and low DNA damage, offering a simple one-step sperm selection device with potential for application in human and animal assisted reproduction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/673619v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@b07026org.highwire.dtl.DTLVardef@192e5b5org.highwire.dtl.DTLVardef@127095aorg.highwire.dtl.DTLVardef@1d70ceb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Crossing mucosal barriers is a central challenge for oral drug delivery, where nanoparticle design must balance stability with mobility in complex fluids. Here, we demonstrate DNA origami as a programmable platform to investigate these processes. Using FRET analysis, we show that DNA nanostructures retain their structural integrity for extended periods in porcine intestinal fluid and mucus, establishing their suitability for biologically relevant environments. Building on this, we used single-particle tracking to assess enzyme-powered propulsion within mucus. Both urease and catalase enhanced diffusion only when anchored to the DNA origami structure, with propulsion persisting for tens of minutes. Importantly, enzyme spatial organization dictated performance: symmetric urease placement improved mobility via uniform local pH shifts, while asymmetric catalase placement enabled efficient bubble-driven propulsion. These results highlight DNA origami as a uniquely versatile tool to dissect structure-function relationships in mucus transport and provide design principles for next-generation, enzyme-powered oral delivery systems.

Delivering biomolecules into pollen tubes that deliver sperm cells for plant fertilization remains technically challenging due to thick cell walls and rapid polarized growth, hindering reproductive engineering. DNA nanotechnology offers a promising alternative over current delivery methods due to their biocompatibility, programmable design, low cytotoxicity, and stimulus-responsive properties, yet their application in plants remains underexplored. Here, we provide the first demonstration of tetrahedral DNA nanostructures (TDNs) as nanocarriers for active, endocytosis-mediated uptake into Arabidopsis pollen tubes, enabling spermidine delivery that shortens pollen tube elongation through actin reorganization and ROS modulation. TDN-treated pollen tubes grew through the Arabidopsis stigma and style, underwent capacitation, and maintained attraction to ovules in a semi-in-vivo assay, preserving reproductive fitness. Furthermore, we demonstrate that functionalization of TDNs with nuclear localization signal peptide significantly enhances nuclear localization. Collectively, these findings establish DNA nanostructures as effective nanocarriers for targeted biomolecule delivery and precise pollen tube modulation, advancing crop reproductive engineering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/710033v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@a09c01org.highwire.dtl.DTLVardef@623943org.highwire.dtl.DTLVardef@9d954corg.highwire.dtl.DTLVardef@1b4c35b_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Cell shape and function are intimately linked, in a way that is mediated by the forces exerted between cells and their environment. The relationship between cell shape and forces has been extensively studied for cells seeded on flat 2-dimensional (2D) substrates, but not for cells in more physiological three-dimensional (3D) settings. Here, we demonstrate a technique called 3D micropatterned traction force microscopy (3D-TFM) to confine cells in three-dimensional wells of defined shape, while simultaneously measuring the forces transmitted between cells and their microenvironment. This technique is based on the 3D micropatterning of polyacrylamide wells and on the calculation of 3D traction force from their deformation. With 3D-TFM, we show that MCF10A breast epithelial cells exert defined, reproducible patterns of forces on their microenvironment, which can be both contractile and extensile. We further show that cells switch from a global contractile to extensile behaviour as their volume is reduced. Our technique enables the quantitative study of cell mechanobiology with full access to 3D cellular forces while having accurate control over cell morphology and the mechanical conditions of the microenvironment.

Stereochemistry plays a crucial role in how molecules interact with complex physiological environments, affecting pharmacokinetics, pharmacodynamics, efficacy, and toxicity. Although these effects are well studied for small-molecular drugs, they are largely overlooked for supramolecular assemblies used in drug delivery. Even for lipid nanoparticles (LNPs)--the most advanced RNA delivery platform--stereochemical effects are rarely investigated and, when considered, are typically limited to the ionizable lipid rather than the overall stereochemical identity of the LNP. Here we separate the ionizable lipid cKK-E12 into its two stereoisomers (trans: R,S/S,R; cis: R,R/S,S), which are normally used as a mixture. LNPs containing the cis isomer exhibit improved physicochemical properties, stability, and protein expression. By systematically varying the stereochemistry of the ionizable lipid, phospholipid, and cholesterol, we reveal stereochemistry-dependent differences in uptake and protein expression across six cell lines and in vivo in zebrafish embryos and mice. AI-assisted cryo-TEM analysis and SAXS link enhanced protein expression to structural differences, demonstrating control over internal lipid phases (lamellar and inverse hexagonal), influencing sample uniformity, and identifying stereochemical identity as a key determinant of functional RNA delivery.

Membrane models of scaffolded discoidal lipid bilayers called nanodiscs have proven to be a valuable tool for the study of membrane proteins in a native environment. DNA-scaffolded membrane model has emerged as an alternative tool for membrane protein studies. Taking advantage of the designability of DNA nanostructure, we created a double-decker double-stranded DNA ring (DDring) to self-assemble DNA-based nanodiscs (DNA-ND). The DDring is 17 nm wide and 4 nm high, and equipped with 28 alkyl chains on the inside that can interact with each hydrophobic leaflet of the lipid bilayer. We further demonstrate the functionality of DNA-ND membrane model with the assembly of membrane proteins. DDrings are suited to neutral or cationic charged phospholipids and detergents. This study provides more insights into the potential use of DNA- assisted nanodiscs for membrane protein characterization.

Copper chalcogenide nanocrystals (NCs) are promising candidates for biophotonic applications due to their tunable optical properties. Concrete methods to examine the relationship between their degradation and toxicity are necessary to enable development of nanoconstructs with reduced toxicity. This study compares the degradation and acute cytotoxicity of three compositions of micelle-coated copper chalcogenide NCs: the fluorescent semiconductor copper indium sulfide (CuInS2), and the plasmonic semiconductors copper sulfide (Cu2-xS) and chalcopyrite copper iron sulfide (CuFeS2). We developed a quantitative degradation assay to assess ion release from these ultra-small nanocrystals, revealing that while all three particles biodegrade, CuInS2 and CuFeS2 undergo rapid degradation in artificial lysosomal fluid, leading to a burst release of indium and iron ions. In cellular toxicity assays, CuInS2 exhibited significantly higher acute cytotoxicity than Cu2-xS and CuFeS2, primarily due to indium-induced necrosis. To mitigate this toxicity, an alternative surface-binding polymer coating was introduced, effectively reducing both the degradation rate and cytotoxicity of CuInS2. These findings highlight the influence of both nanocrystal composition and coating chemistry in moderating the acute cytotoxity of degradable nanocrystals, demonstrating that tuning of composition and degradation rate can be used to moderate nanoparticle toxicity.

DNA origami, a method of folding DNA into precise nanostructures, has emerged as a powerful tool to design complex nanoscale shapes with movable parts. DNA origami has great potential as a drug delivery system that can encapsulate and protect a range of cargos spanning small molecules through large proteins, while remaining stable in a variety of ex vivo processing conditions and in vivo environments. DNA origami has been utilized for drug delivery applications, but the vast majority of these structures have been flexible, flat 2D or solid 3D nanostructures. There is a crucial need for a hollow and completely enclosed design capable of holding any type of cargo. In this paper, we present the design and assembly of a hollow DNA origami "box" with two actuatable lids. We characterize isothermal conditions for structural assembly in minutes that eliminates the need for a thermocycler. The stability of these structures is outstanding, remaining stable at body temperature and low pH for weeks and in the presence of solvents and biological fluids over several days. We demonstrate that passive loading of small molecules is charge dependent. We also outline an approach to design staple extensions pointing into the cavity or outside of the hollow DNA origami, allowing for either active loading of protein or the potential for decoration with passivating or targeting molecules. Future work includes fitting this hollow DNA origami structure with alternative lid opening mechanisms to release a variety of different cargos in response to environmental cues. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/586853v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@97179corg.highwire.dtl.DTLVardef@18f0b46org.highwire.dtl.DTLVardef@2d390eorg.highwire.dtl.DTLVardef@fcf35c_HPS_FORMAT_FIGEXP M_FIG C_FIG