DNA nanotechnology has made initial progress toward developing gene-encoded DNA origami nanoparticles (NPs) that display potential utility for future gene therapy applications. However, due to the challenges involved with gene delivery into cells including transport through the membrane, intracellular targeting, and inherent expression of nucleases along with interference from other active proteins, it can be difficult to more directly study the effect of DNA NP design on subsequent gene expression. In this work, we demonstrate an approach for studying the expression of gene-encoding DNA origami NPs without the use of cells. We utilize a pure E. coli-derived cell-free transcription-translation (TXTL) system, which is composed of optimized components from bacterial expression, for benchtop studies to assess how the promoter sequence in conjunction with structural design of the DNA NP template affects gene expression. The gene for an optimized Renilla luciferase was first amplified into a single-stranded (ss) scaffold strand and then folded into a 12-helix bundle DNA NP with exogenous staple strands as a test platform. Using luciferase-based bioluminescence assays to characterize the relative protein expression level, it was found that the gene can still be transcribed when folded, albeit at a lower rate than the double-stranded DNA gene segment. On comparing three variants of DNA NP with different promoter configurations, results indicate that a promoter designed to remain in ssDNA form has reduced protein expression from the DNA NP, and replacing the promoter sequence with an arbitrary sequence significantly lowers protein expression. This work demonstrates the power inherent in cell-free TXTL systems as an aid to study the gene expression capabilities of DNA NPs toward design and development of future applications.

The physical and chemical properties of synthetic DNA have transformed this prototypical biopolymer into a versatile nanoscale building block material in the form of DNA nanotechnology. DNA nanotechnology is, in turn, providing unprecedented precision bioengineering for numerous biomedical applications at the nanoscale including next generation immune-modulatory materials, vectors for targeted delivery of nucleic acids, drugs, and contrast agents, intelligent sensors for diagnostics, and theranostics, which combines several of these functionalities into a single construct. Assembling a DNA nanostructure to be programmed with a specific number of targeting moieties on its surface to imbue it with concomitant cellular uptake and retention capabilities along with carrying a specific therapeutic dose is now eminently feasible due to the extraordinary self-assembling properties and high formation efficiency of these materials. However, what remains still only partially addressed is how exactly this class of materials is taken up into cells in both the native state and as targeted or chemically facilitated, along with how stable they are inside the cellular cytosol and other cellular organelles. In this minireview, we summarize what is currently reported in the literature about how (i) DNA nanostructures are taken up into cells along with (ii) what is understood about their subsequent stability in the complex multi-organelle environment of the cellular milieu along with biological fluids in general. This allows us to highlight the many challenges that still remain to overcome in understanding DNA nanostructure-cellular interactions in order to fully translate these exciting new materials.

In a previous study, we demonstrated a novel manufacturing approach to fabricate multi-channel scaffolds (MCS) for use in spinal cord injuries (SCI). In the present study, we extended similar materials processing technology to fabricate significantly longer (5X) porous poly caprolactone (PCL) MCS and evaluated their efficacy in 1 cm sciatic peripheral nerve injury (PNI) model. Due to the increase in MCS dimensions and the challenges that may arise in a longer nerve gap model, microstructural characterization involved MCS wall permeability to assess nutrient flow, topography, and microstructural uniformity to evaluate the potential for homogeneous linear axon guidance. It was determined that the wall permeability dramatically varied from 0.02 ± 0.01 × 10-13 to 21.7 ± 11.4 × 10-13 m2 for 50% and 70% porous PCL, respectively. Using interferometry, the porous PCL surface roughness was determined to be 10.7 ± 1.2 μm, which is believed to be sufficient to promote cell integration. Using micro computed tomography, the 3D MCS microstructure was determined to be uniform over 1 cm with an open lumen volume of 44.6% ± 3.6%. In vivo implantation, in the rat sciatic nerve model, over 4 weeks, demonstrated that MCS scaffolds maintained structural integrity, were biocompatible, and supported linear axon guidance and distal end egress over 1 cm. Taken together, this study demonstrated that MCS technology previously developed for the SCI is also relevant to longer nerve gap PNI.